Method of improving the movement of a target polynucleotide with respect to a transmembrane pore

ABSTRACT

The invention relates to improving the movement of a target polynucleotide with respect to a transmembrane pore when the movement is controlled by a polynucleotide binding protein. The invention also relates to improved transmembrane pores and polynucleotide binding proteins.

This Application is a continuation of U.S. application Ser. No.15/308,252, filed on Nov. 1, 2016, which is a national stage filingunder 35 U.S.C. § 371 of PCT International Application No.PCT/GB2015/051291, which has an international filing date of May 1,2015, and claims foreign priority benefits under 35 U.S.C. § 119(a)-(d)or 35 U.S.C. § 365(b) of British application number 1417708.3, filedOct. 7, 2014, British application number 1417712.5, filed Oct. 7, 2014,and British application number 1407809.1, filed May 2, 2014, thecontents of each of which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to improving the movement of a targetpolynucleotide with respect to a transmembrane pore when the movement iscontrolled by a polynucleotide binding protein. The invention alsorelates to improved transmembrane pores and polynucleotide bindingproteins.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe strand sequencing method, a single polynucleotide strand is passedthrough the pore and the identities of the nucleotides are derived.Strand sequencing can involve the use of a polynucleotide bindingprotein to control the movement of the polynucleotide through the pore.

The different forms of Msp are porins from Mycobacterium smegmatis. MspAis a 157 kDa octameric porin from Mycobacterium smegmatis. Wild-typeMspA does not interact with DNA in a manner that allows the DNA to becharacterised or sequenced. The structure of MspA and the modificationsrequired for it to interact with and characterise DNA have been welldocumented (Butler, 2007, Nanopore Analysis of Nucleic Acids, Doctor ofPhilosophy Dissertation, University of Washington; Gundlach, Proc NatlAcad Sci USA. 2010 Sep. 14; 107(37):16060-5. Epub 2010 Aug. 26; andInternational Application No. PCT/GB2012/050301 (published asWO/2012/107778).

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that polynucleotide bindingprotein-controlled movement of a target polynucleotide with respect to atransmembrane pore is improved by modifying a part of the transmembranepore which interacts with the polynucleotide binding protein and/or apart of the polynucleotide binding protein which interacts with thetransmembrane pore.

Accordingly, the invention provides a method of improving the movementof a target polynucleotide with respect to a transmembrane pore when themovement is controlled by a polynucleotide binding protein, comprisingmodifying a part of the transmembrane pore which interacts with thepolynucleotide binding protein and/or a part of the polynucleotidebinding protein which interacts with the transmembrane pore and therebyimproving the movement of the target polynucleotide with respect to thetransmembrane pore.

The invention also provides:

a method of moving a target polynucleotide with respect to atransmembrane pore using a polynucleotide binding protein, comprising

a) providing a transmembrane pore and a polynucleotide binding proteinin which a part of the transmembrane pore which interacts with thepolynucleotide binding protein and/or a part of the polynucleotidebinding protein which interacts with the transmembrane pore has beenmodified; and

b) contacting the transmembrane pore and polynucleotide binding proteinprovided in a) with the target polynucleotide such that the proteincontrols the movement of the polynucleotide with respect to thetransmembrane pore;

a method of characterising a target polynucleotide, comprising:

a) providing a transmembrane pore and a polynucleotide binding proteinin which a part of the transmembrane pore which interacts with thepolynucleotide binding protein and/or a part of the polynucleotidebinding protein which interacts with the transmembrane pore has beenmodified;

b) contacting the transmembrane pore and polynucleotide binding proteinprovided in (a) with the target polynucleotide such that the proteincontrols the movement of the polynucleotide with respect to thetransmembrane pore; and

c) taking one or more measurements as the polynucleotide moves withrespect to the transmembrane pore, wherein the measurements areindicative of one or more characteristics of the polynucleotide, andthereby characterising the target polynucleotide;

a transmembrane pore in which a part of the transmembrane pore whichinteracts with a polynucleotide binding protein has been modified;

a mutant Msp monomer comprising a variant of SEQ ID NO: 2 in which apart of the monomer which interacts with a polynucleotide bindingprotein has been modified;

a construct comprising two or more covalently attached MspA monomers,wherein at least one of the monomers is a mutant monomer of theinvention;

a homo-oligomeric pore derived from Msp comprising identical mutantmonomers of the invention or identical constructs of the invention;

a hetero-oligomeric pore derived from Msp comprising at least one mutantmonomer of the invention or at least one construct of the invention;

a polynucleotide binding protein in which a part of the protein whichinteracts with a transmembrane pore has been modified;

a combination of a transmembrane pore and a polynucleotide bindingprotein in which a part of the transmembrane pore which interacts withthe polynucleotide binding protein and/or a part of the polynucleotidebinding protein which interacts with the transmembrane pore has beenmodified;

a kit for characterising a target polynucleotide comprising (a) atransmembrane pore of the invention and (b) the components of amembrane;

a kit for characterising a target polynucleotide comprising (a) apolynucleotide binding protein of the invention and (b) a polynucleotideadaptor to which the polynucleotide binding protein is optionally bound;

an apparatus for characterising target polynucleotides in a sample,comprising (a) a plurality of transmembrane pores of the invention or aplurality of combinations of the invention and (b) a plurality ofmembranes; and

a method of characterising a target polynucleotide, comprising:

a) providing a transmembrane pore and a polymerase in which a part ofthe transmembrane pore which interacts with the polymerase and/or a partof the polymerase which interacts with the transmembrane pore has beenmodified;

b) contacting the target polynucleotide with the transmembrane pore andpolymerase provided in a) and labelled nucleotides such that phosphatelabelled species are sequentially added to the target polynucleotide bythe polymerase, wherein the phosphate species contain a label specificfor each nucleotide; and

c) detecting the phosphate labelled species using the transmembrane poreand thereby characterising the polynucleotide.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the three different initial simulation orientations of T4Dda-E94C/A360C/C109A/C136A (SEQ ID NO: 24 with mutationsE94C/A360C/C109A/C136A and then (ΔM1)G1G2) with respect to eitherMspA-(G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8 (SEQ ID NO: 2with mutations G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K=MspAmutant 1) orMspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119=MspA mutant 2). Thedifference between run 1 and run 2 was that both the enzyme and pore haddifferent side chain conformations despite the pore and enzyme being inthe same position. In run three the enzyme has been tilted slightly withrespect to the nanopore.

FIG. 2 shows a plot (y-axis label=number of pore/enzyme contacts, x-axislabel=pore amino acid residue number) of the interaction points of thenanopore MspA mutant 1 with T4 Dda-E94C/A360C/C109A/C136A. Each row ofthe plot shows the interaction points for the different enzyme/nanoporeorientations e.g. runs 1-3.

FIG. 3 shows a plot (y-axis label=number of pore/enzyme contacts, x-axislabel=enzyme amino acid residue number) of the interaction points of theenzyme T4 Dda-E94C/A360C/C109A/C136A with MspA mutant 1. Each row of theplot shows the interaction points for the different enzyme/nanoporeorientations e.g. runs 1-3.

FIG. 4 shows a plot (y-axis label=number of pore/enzyme contacts, x-axislabel=pore amino acid residue number) of the interaction points of thenanopore MspA mutant 2 with T4 Dda-E94C/A360C/C109A/C136A. Each row ofthe plots shows the interaction points for the different enzyme/nanoporeorientations e.g. runs 1-3.

FIG. 5 shows a plot (y-axis label=number of pore/enzyme contacts, x-axislabel=enzyme amino acid residue number) of the interaction points of theenzyme T4 Dda-E94C/A360C/C109A/C136A with MspA mutant 2. Each row of theplot shows the interaction points for the different enzyme/nanoporeorientations e.g. runs 1-3.

FIG. 6 (A) shows two regions of a plot (y-axis label=pore amino acidresidue number, x-axis label=enzyme amino acid residue number) whichshows which amino acids in the pore (MspA mutant 2) interact withparticular amino acids in the enzyme (T4 Dda-E94C/A360C/C109A/C136A)from run 1. FIG. 6 (B) shows a region of a plot (y-axis label (a1)=poreamino acid residue number, y-axis label (a2)=number of pore/enzymecontacts, x-axis label=enzyme amino acid residue number) which showswhich amino acids in the pore (MspA mutant 2) interact with particularamino acids in the enzyme (T4 Dda-E94C/A360C/C109A/C136A) from run 3.The grey bands in the plots indicate an interaction between amino acids.The darkness of the grey band corresponds to the number of interactionsbetween enzyme/pore, with dark grey=many interactions and lightgrey=fewer interactions. The first amino acid in each box corresponds tothe interacting amino acid in the MspA mutant 2 and the second aminoacid corresponds to the interacting amino acids in T4Dda-E94C/A360C/C109A/C136A.

FIG. 7 shows DNA construct X used in Example 2. Section A correspondedto thirty iSpC3 spacers. Section B corresponded to SEQ ID NO: 28. LabelC corresponded to the enzyme T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO:24 with mutations E94C/C109A/C136A/A360C). Section D corresponded tofour iSp18 spacers. Section E corresponded to SEQ ID NO: 29. Section Fcorresponded to four iSNitInd groups (IDT). Section G corresponded toSEQ ID NO: 30. Section H corresponded to four iSpC3 spacers. Section Jcorresponded to SEQ ID NO: 31. Section K corresponded to SEQ ID NO: 32.Section L corresponded to six iSp18 spacers and two thymine residues.Section M corresponded to a 3′ cholesterol tether.

FIG. 8 shows DNA construct Y used in Example 2. Section A correspondedto SEQ ID NO: 33. Label B corresponded to the enzyme T4Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutationsE94C/C109A/C136A/A360C). Section C corresponded to four iSpC3 spacers.Section D corresponded to SEQ ID NO: 27. Section E corresponded to fouri5NitInd groups (IDT). Section F corresponded to SEQ ID NO: 34. SectionG corresponded to SEQ ID NO: 32. Section H corresponded to six iSp18spacers and two thymine residues. Section I corresponded to a 3′cholesterol tether.

FIG. 9 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56W/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56W/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 10 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)E59Y/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59Y/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119). Sections B and C showzoomed in regions of current trace A.

FIG. 11 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 12 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56Y/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations)D56Y/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 13 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E57D/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsD56N/E57D/E59R/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of theamino acids L74/G75/D118/L119). Sections B and C show zoomed in regionsof current trace A.

FIG. 14 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59T/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59T/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 15 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59Q/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59Q/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 16 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59F/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119). Sections B and C showzoomed in regions of current trace A.

FIG. 17 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 18 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 19 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134N/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134N/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 20 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59W/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59W/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 21 shows the three different initial simulation orientations ofPhi29 DNA polymerase-(D12A/D66A) (SEQ ID NO: 9 with mutations D12A/D66A)with respect to αHL-(E111N/K147N)8 (SEQ ID NO: 4). The differencebetween run 2 and run 3 was that both the enzyme and pore had differentside chain conformations despite the pore and enzyme being in the sameposition. In run one the enzyme has been tilted slightly with respect tothe nanopore.

FIG. 22 shows a plot (y-axis label=number of pore/enzyme contacts,x-axis label=pore amino acid residue number) of the interaction pointsof the nanopore αHL-(E111N/K147N)8 with Phi29 DNApolymerase-(D12A/D66A). Each row of the plot shows the interactionpoints for the different enzyme/nanopore orientations e.g. runs 1-3.

FIG. 23 shows a plot (y-axis label=number of pore/enzyme contacts,x-axis label=enzyme amino acid residue number) of the interaction pointsof the enzyme Phi29 DNA polymerase-(D12A/D66A) with αHL-(E111N/K147N)8.Each row of the plot shows the interaction points for the differentenzyme/nanopore orientations e.g. runs 1-3.

FIG. 24 shows a zoomed in region of a plot (y-axis label (a1)=pore aminoacid residue number, y-axis label (a2)=number of pore/enzyme contacts,x-axis label=enzyme amino acid residue number) which shows which aminoacids in the pore (αHL-(E111N/K147N)8) interact with particular aminoacids in the enzyme (Phi29 DNA polymerase-(D12A/D66A)) from run 1. Thegrey bands in the plot indicate an interaction between amino acids. Thedarkness of the grey band corresponds to the number of interactionsbetween enzyme/pore, with dark grey=many interactions and lightgrey=fewer interactions. The first amino acid in each box corresponds tothe interacting amino acid in the αHL-(E111N/K147N)8 and the secondamino acid corresponds to the interacting amino acids in Phi29 DNApolymerase-(D12A/D66A).

FIG. 25 shows a zoomed in region of a plot (y-axis label (a1)=pore aminoacid residue number, y-axis label (a2)=number of pore/enzyme contacts,x-axis label=enzyme amino acid residue number) which shows which aminoacids in the pore (αHL-(E111N/K147N)8) interact with particular aminoacids in the enzyme (Phi29 DNA polymerase-(D12A/D66A)) from run 1. Theblack bands in the plot indicate an interaction between amino acids. Thedarkness of the grey band corresponds to the number of interactionsbetween enzyme/pore, with dark grey=many interactions and lightgrey=fewer interactions. The first amino acid in each box corresponds tothe interacting amino acid in the αHL-(E111N/K147N)8 and the secondamino acid corresponds to the interacting amino acids in Phi29 DNApolymerase-(D12A/D66A).

FIG. 26 shows two zoomed in regions of a plot (y-axis label (a1)=poreamino acid residue number, y-axis label (a2)=number of pore/enzymecontacts, x-axis label=enzyme amino acid residue number) which showswhich amino acids in the pore (αHL-(E111N/K147N)8 interact withparticular amino acids in the enzyme (Phi29 DNA polymerase-(D12A/D66A)from run 2. The grey bands in the plot indicate an interaction betweenamino acids. The darkness of the grey band corresponds to the number ofinteractions between enzyme/pore, with dark grey=many interactions andlight grey=fewer interactions. The first amino acid in each boxcorresponds to the interacting amino acid in the αHL-(E111N/K147N)8 andthe second amino acid corresponds to the interacting amino acids inPhi29 DNA polymerase-(D12A/D66A).

FIG. 27 shows a zoomed in region of a plot (y-axis label (a1)=pore aminoacid residue number, y-axis label (a2)=number of pore/enzyme contacts,x-axis label=enzyme amino acid residue number) which shows which aminoacids in the pore (αHL-(E111N/K147N)8 interact with particular aminoacids in the enzyme (Phi29 DNA polymerase-(D12A/D66A)) from run 2. Thegrey bands in the plot indicate an interaction between amino acids. Thedarkness of the grey band corresponds to the number of interactionsbetween enzyme/pore, with dark grey=many interactions and lightgrey=fewer interactions. The first amino acid in each box corresponds tothe interacting amino acid in the αHL-(E111N/K147N)8 and the secondamino acid corresponds to the interacting amino acids in Phi29 DNApolymerase-(D12A/D66A).

FIG. 28 shows a zoomed in region of a plot (y-axis label (a1)=pore aminoacid residue number, y-axis label (a2)=number of pore/enzyme contacts,x-axis label=enzyme amino acid residue number) which shows which aminoacids in the pore (αHL-(E111N/K147N)8 interact with particular aminoacids in the enzyme (Phi29 DNA polymerase-(D12A/D66A) from run 3. Thegrey bands in the plot indicate an interaction between amino acids. Thedarkness of the grey band corresponds to the number of interactionsbetween enzyme/pore, with dark grey=many interactions and lightgrey=fewer interactions. The first amino acid in each box corresponds tothe interacting amino acid in the αHL-(E111N/K147N)8 and the secondamino acid corresponds to the interacting amino acids in Phi29 DNApolymerase-(D12A/D66A).

FIG. 29 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 30 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 31 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/K199L/A360C) controlled the translocation of theDNA construct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 32 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56L/E59L/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56L/E59L/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 33 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)G1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsG1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of the aminoacids L74/G75/D118/L119). Sections B and C show zoomed in regions ofcurrent trace A.

FIG. 34 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/K199L/A360C) controlled the translocation of theDNA construct X through the MspA nanopore MspA-((Del-L74/G75/D118/L119)G1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8 (SEQ ID NO: 2 withmutations G1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K and deletion ofthe amino acids L74/G75/D118/L119). Sections B and C show zoomed inregions of current trace A.

FIG. 35 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda-E94C/C109A/C136A/A360C) controlled the translocation of the DNAconstruct X through the MspA nanoporeMspA-((Del-L74/G75/D118/L119)D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Sections B and Cshow zoomed in regions of current trace A.

FIG. 36 shows a bar chart of various enzyme/pore combinations which wereinvestigated in order to determine the number of slips forward perkilobase and the % bases missed when construct X was translocatedthrough the nanopore under the control of the enzyme (x-axis label=poreand enzyme combinations 1-7 (see table 12) and y-axis label A=slipsforward per kilobase and y-axis label B=% bases missed). The bar'slabelled with a black star correspond to the % bases missed and thoselabelled with a circle correspond to the slips forward per kilobase.

FIG. 37 shows a cartoon representation of the wild-type MspA nanopore.Region 1 corresponds to the cap forming region and includes residues1-72 and 122-184. Region 2 corresponds to the barrel forming region andincludes residues 73-82 and 112-121. Region 3 corresponds to theconstriction and loops region and includes residues 83-111.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe wild-type MspA monomer. This mutant lacks the signal sequence.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of thewild-type MspA monomer. This mutant lacks the signal sequence.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNApolymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′ 5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(http://www.neb.com/nebecomm/products/productM0262.asp). Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of Hel308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of Hel308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of Hel308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows positions 72 to 82 of SEQ ID NO: 2.

SEQ ID NO: 27 shows positions 111 to 121 of SEQ ID NO: 2.

SEQ ID NO: 28 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 29 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 30 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 31 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 32 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 33 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 34 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 35 shows the polynucleotide sequence encoding the lyseninmonomer.

SEQ ID NO: 36 shows the amino acid sequence of the lysenin monomer.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “apolynucleotide binding protein includes two or more such proteins,reference to “a helicase” includes two or more helicases, reference to“a monomer” refers to two or more monomers, reference to “a pore”includes two or more pores and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Modification Methods

The present invention provides a method of improving the movement of atarget polynucleotide with respect to a transmembrane pore when themovement is controlled by a polynucleotide binding protein. The methodis preferably for improving the movement of a target polynucleotidethrough a transmembrane pore when the movement is controlled by apolynucleotide binding protein. Target polynucleotides are discussed inmore detail below.

The method comprises modifying a part of the transmembrane pore whichinteracts with the polynucleotide binding protein or a part of thepolynucleotide binding protein which interacts with the transmembranepore. The method may comprise modifying both the transmembrane pore andthe polynucleotide binding protein.

Methods of modifying pores and proteins, such as via amino acidintroductions and/or substitutions, are known in the art and arediscussed in more detail below.

Characterisation, such as sequencing, of a polynucleotide using atransmembrane pore typically involves analysing polymer units made up ofk nucleotides where k is a positive integer (i.e. ‘k-mers’). This isdiscussed in International Application No. PCT/GB2012/052343 (publishedas WO 2013/041878). As the target polynucleotide moves with respect to,or through the pore, different k-mers within the polynucleotide areanalysed, typically by measuring the current flowing through the pore.The movement of the polynucleotide with respect to, such as through, thepore can be viewed as movement from one k-mer to another or from k-merto k-mer.

The method of the invention preferably provides more consistent movementof the target polynucleotide with respect to, such as through, thetransmembrane pore. The method preferably provides more consistentmovement from one k-mer to another or from k-mer to k-mer as the targetpolynucleotide moves with respect to, such as through, the pore. Themethod preferably allows the target polynucleotide to move with respectto, such as through, the transmembrane pore more smoothly. The methodpreferably provides more regular or less irregular movement of thetarget polynucleotide with respect to, such as through, thetransmembrane pore.

The method preferably reduces the amount of slipping forward associatedwith the movement of the target polynucleotide with respect to, such asthrough, the pore. Some helicases including the Dda helicase used in theExample move along the polynucleotide in a 5′ to 3′ direction. When the5′ end of the polynucleotide (the end away from which the helicasemoves) is captured by the pore, the helicase works with the direction ofthe field resulting from the applied potential and moves the threadedpolynucleotide into the pore and into the trans chamber. Slippingforward involves the DNA moving forwards relative to the pore (i.e.towards its 3′ and away from it 5′ end) at least 4 consecutivenucleotides and typically more than 10 consecutive nucleotides. Slippingforward may involve movement forward of 100 consecutive nucleotides ormore and this may happen more than once in each strand.

The method of the invention preferably reduces the noise associated withthe movement of the target polynucleotide with respect to, such asthrough, the transmembrane pore. Unwanted movement of the targetpolynucleotide in any dimension as a k-mer is being analysed typicallyresults in noise in the current signature or level for the k-mer. Themethod of the invention may reduce this noise by reducing unwantedmovement associated with one or more k-mers, such as each k-mer, in thetarget polynucleotide. The method of the invention may reduce the noiseassociated with the current level or signature for one or more k-mers,such as each k-mer, in the target polynucleotide.

In a preferred embodiment, the target polynucleotide is double strandedand the method reduces the noise associated with the movement of thecomplement strand to a greater degree than it reduces the noiseassociated with the movement of the template strand and/or the methodincreases the consistency of the movement of the complement strand to agreater degree than it increases the consistency of the movement of thetemplate strand. This is advantageous for strand sequencing of doublestranded target polynucleotides. The two stands of the double strandedpolynucleotide are preferably linked by a bridging moiety, such as ahairpin loop or hairpin loop adaptor. This is discussed in more detailbelow.

Transmembrane Pore

A transmembrane pore is a structure that crosses the membrane to somedegree. It permits hydrated ions driven by an applied potential to flowacross or within the membrane. The transmembrane pore typically crossesthe entire membrane so that hydrated ions may flow from one side of themembrane to the other side of the membrane. However, the transmembranepore does not have to cross the membrane. It may be closed at one end.For instance, the pore may be a well, gap, channel, trench or slit inthe membrane along which or into which hydrated ions may flow.

Any transmembrane pore may be used in the invention. The pore may bebiological or artificial. Suitable pores include, but are not limitedto, protein pores, polynucleotide pores and solid state pores. The poremay be a polynucleotide origami pore, such as a DNA origami pore(Langecker et al., Science, 2012; 338: 932-936).

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as polynucleotides, toflow from one side of a membrane to the other side of the membrane. Inthe present invention, the transmembrane protein pore is capable offorming a pore that permits hydrated ions driven by an applied potentialto flow from one side of the membrane to the other. The transmembraneprotein pore preferably permits analytes such as nucleotides orpolynucleotides to flow from one side of the membrane, such as atriblock copolymer membrane, to the other. The transmembrane proteinpore typically allows a polynucleotide, such as DNA or RNA, to be movedthrough the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as at least 6,at least 7, at least 8 or at least 9 subunits. The pore is preferably ahexameric, heptameric, octameric or nonameric pore. The pore may be ahomo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β pore forming toxins,such as α-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F(OmpF), outer membrane porin G (OmpG), outer membrane phospholipase Aand Neisseria autotransporter lipoprotein (NalP), and other pores, suchas lysenin. α-helix bundle pores comprise a barrel or channel that isformed from α-helices. Suitable α-helix bundle pores include, but arenot limited to, inner membrane proteins and a outer membrane proteins,such as WZA and ClyA toxin. The transmembrane pore may be derived fromlysenin. Suitable pores derived from lysenin are disclosed inInternational Application No. PCT/GB2013/050667 (published as WO2013/153359). The transmembrane pore may be derived from Msp, such asMspA, or from α-hemolysin (α-HL).

The unmodified transmembrane pore used in the invention preferablycomprises seven or more monomers comprising the sequence shown in SEQ IDNO: 2 or a variant thereof. The unmodified transmembrane pore morepreferably comprises 8 or 9 monomers comprising the sequence shown inSEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 and variants thereof arediscussed in more detail below. The pores modified in accordance withthe invention may comprise any of the variants discussed below,especially the variants described with reference to the mutant Mspmonomers of the invention.

In SEQ ID NO: 2 or a variant thereof, the part of the transmembrane porewhich interacts with the polynucleotide binding protein typicallycomprises the amino acids at positions 12, 14, 48, 52, 53, 54, 55, 56,57, 58, 59, 60, 134, 135, 136, 137, 138, 139, 169 and 170. These numberscorrespond to the relevant positions in SEQ ID NO: 2 and may need to bealtered in the case of variants where one or more amino acids have beeninserted or deleted compared with SEQ ID NO: 2. A skilled person iscapable of determining the corresponding position in a variant of SEQ IDNO: 2. For instance, position 4 in SEQ ID NO: 2 becomes position 6 in avariant having two amino acids added at the amino (N) terminus. If thevariant is formed only by substitution of amino acids in SEQ ID NO: 2(i.e. no amino acids are added to or deleted from SEQ ID NO: 2), thecorresponding positions in the variant typically have the same numberingat the positions in SEQ ID NO: 2. The same is true for SEQ ID NOs: 4, 9,24 and 36.

The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises the amino acids atpositions:

(a) 12, 14, 52, 54, 56, 57, 59, 134, 136, 138, 139 and 169 in SEQ ID NO:2 or at the corresponding positions in a variant thereof;

(b) 12, 14, 56, 57, 59, 134, 136, 139 and 169 in SEQ ID NO: 2 or at thecorresponding positions in a variant thereof;

(c) 56, 57, 59, 134, 136, 139 and 169 in SEQ ID NO: 2 or at thecorresponding positions in a variant thereof; or

(d) 56, 57, 59, 134 and 139 in SEQ ID NO: 2 or at the correspondingpositions in a variant thereof.

The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises the amino acid atposition 56 in SEQ ID NO: 2 or at the corresponding position in thevariant thereof. The amino acid at position 56 (aspartic acid; D) may bereplaced with asparagine (N), arginine (R), phenylalanine (F), tyrosine(Y) or leucine (L).

The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises the amino acid atposition 59 in SEQ ID NO: 2 or at the corresponding position in avariant thereof. The amino acid at position 59 (glutamic acid; E) may bereplaced with asparagine (N), arginine (R), phenylalanine (F), tyrosine(Y) or leucine (L).

The transmembrane protein pore may also be derived from α-hemolysin(α-HL). The wild type α-HL pore is formed of seven identical monomers orsubunits (i.e. it is heptameric). The sequence of one monomer or subunitof α-hemolysin-NN is shown in SEQ ID NO: 4. The transmembrane proteinpore preferably comprises seven monomers each comprising the sequenceshown in SEQ ID NO: 4 or a variant thereof. Amino acids 1, 7 to 21, 31to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to242, 262 to 265, 272 to 274, 287 to 290 and 294 of SEQ ID NO: 4 formloop regions. Residues 113 and 147 of SEQ ID NO: 4 form part of aconstriction of the barrel or channel of α-HL.

In such embodiments, a pore comprising seven proteins or monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof arepreferably used in the method of the invention. The seven proteins maybe the same (homo-heptamer) or different (hetero-heptamer).

In SEQ ID NO: 4, the part of the transmembrane pore which interacts withthe polynucleotide binding protein typically comprises the amino acidsat positions 16, 17, 18, 19, 21, 46, 47, 93, 236, 237, 238, 239, 240,241, 242, 281, 283, 285, 287, 288 and 293. These numbers correspond tothe relevant positions in SEQ ID NO: 4 and may need to be altered in thecase of variants where one or more amino acids have been inserted ordeleted compared with SEQ ID NO: 4. A skilled person can determine thecorresponding positions in a variant as discussed above.

The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises the amino acids atpositions:

(a) 17, 18, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242, 287, 288and 293 in SEQ ID NO: 4 or at the corresponding positions in the variantthereof;

(b) 17, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242 and 287 in SEQID NO: 4 or at the corresponding positions in the variant thereof; or

(c) 17, 19, 46, 93, 236, 237, 239, 240, 287 and 288 in SEQ ID NO: 4 orat the corresponding positions in the variant thereof.

The amino acids at any of these positions may be replaced withphenylalanine (F), tryptophan (W), isoleucine (I), leucine (L), valine(V), alanine (A), arginine (R), lysine (K), aspartic acid (D), glutamicacid (E) or tyrosine (Y) in accordance with the invention.

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 4 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into an amphiphilic layer, such as a triblock copolymermembrane, along with other appropriate subunits and its ability tooligomerise to form a pore may be determined. Methods are known in theart for inserting subunits into amphiphilic layers, such as triblockcopolymer membranes. Suitable methods are discussed above.

The variant may include modifications that facilitate covalentattachment to or interaction with the polynucleotide binding protein.The variant preferably comprises one or more reactive cysteine residuesthat facilitate attachment to the polynucleotide binding protein. Forinstance, the variant may include a cysteine at one or more of positions8, 9, 17, 18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or on the aminoor carboxy terminus of SEQ ID NO: 4. Preferred variants comprise asubstitution of the residue at position 8, 9, 17, 237, 239 and 287 ofSEQ ID NO: 4 with cysteine (A8C, T9C, N17C, K237C, S239C or E287C). Thevariant is preferably any one of the variants described in InternationalApplication No. PCT/GB09/001690 (published as WO 2010/004273),PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133(published as WO 2010/086603).

The variant may also include modifications that facilitate anyinteraction with nucleotides.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium.Alternatively, the variant may be expressed in vitro or recombinantly bya bacterium such as Escherichia coli. Variants also includenon-naturally occurring variants produced by recombinant technology.Over the entire length of the amino acid sequence of SEQ ID NO: 4, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 4 over the entire sequence. There maybe at least 80%, for example at least 85%, 90%/o or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed below.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 4 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:4 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 4. Such fragments retainpore-forming activity. Fragments may be at least 50, 100, 200 or 250amino acids in length. A fragment preferably comprises the pore-formingdomain of SEQ ID NO: 4. Fragments typically include residues 119, 121,135. 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 4 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 4 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a (β-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 4 typically comprisesthe regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQID NO: 4 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 4 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the β-strand regions ofSEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

The variant of SEQ ID NO: 4 may be modified to assist its identificationor purification as discussed below.

The transmembrane protein pore may also be derived from lysenin. Theunmodified transmembrane pore used in the invention preferably comprisesat least one monomer comprising the sequence shown in SEQ ID NO: 36 or avariant thereof.

In SEQ ID NO: 36 or a variant thereof, the part of the transmembranepore which interacts with the polynucleotide binding protein typicallycomprises the amino acids at positions (i) 31 (serine; S), (ii) 33(serine; S), (iii) 108 (proline; P), (iv) 109 (proline; P), (v) 110(threonine) and (vi) 138 (proline; P). These numbers correspond to therelevant positions in SEQ ID NO: 36 and may need to be altered in thecase of variants where one or more amino acids have been inserted ordeleted compared with SEQ ID NO: 36. A skilled person can determine thecorresponding positions in a variant as discussed above.

The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises any number andcombination of these amino acids in SEQ ID NO: 36 or the variantthereof. The part of the transmembrane pore which interacts with thepolynucleotide binding protein preferably comprises the amino acids atpositions {i}, {ii}, {iii}, {iv}, {v}, {vi}, {i,ii}, {i,iii}, {i,iv},{i,v}, {i,vi}, {ii,iii}, {ii,iv}, {ii,v}, {ii,vi}, {iii,iv}, {iii,v},{iii,vi}, {iv,v}, {iv,vi}, {v,vi}, {i,ii,iii}, {i,ii,iv}, {i,ii,v},{i,ii,vi}, {i,iii,iv}, {i,iii,v}, {i,iii,vi}, {i,iv,v}, {i,iv,vi},{i,v,vi}, {ii,iii,iv}, {ii,iii,v}, {ii,iii,vi}, {ii,iv,v}, {ii,iv,vi},{ii,v,vi}, {iii,iv,v}, {iii,iv,vi}, {iii,v,vi}, {iv,v,vi},{i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iii,vi}, {i,ii,iv,v}, {i,ii,iv,vi},{i,ii,v,vi}, {i,iii,iv,v}, {i,iii,iv,vi}, {i,iii,v,vi}, {i,iv,v,vi},{ii,iii,iv,v}, {ii,iii,iv,vi}, {ii,iii,v,vi}, {ii,iv,v,vi},{iii,iv,v,vi}, {i,ii,iii,iv,v}, {i,ii,iii,iv,vi}, {i,ii,iii,v,vi},{i,ii,iv,v,vi}, {i,iii,iv,v,vi}, {ii,iii,iv,v,vi} or {i,ii,iii,iv,v,vi}in SEQ ID NO: 36 or at the corresponding positions in the variantthereof.

Any number and combination of (i) to (vi) as set out above may bereplaced with phenylalanine (F), tryptophan (W), isoleucine (I), leucine(L), valine (V), alanine (A), arginine (R), lysine (K), aspartic acid(D), glutamic acid (E) or tyrosine (Y) in accordance with the invention.

A variant of SEQ ID NO: 36 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 36 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art as discussed above.

Over the entire length of the amino acid sequence of SEQ ID NO: 36, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 36 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed below.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 36 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:36 may additionally be deleted from the variants described above. Up to1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 36. Such fragments retainpore forming activity. This may be assayed as described above. Fragmentsmay be at least 50, 100, 150, 200 or 250 amino acids in length. Suchfragments may be used to produce the pores of the invention. Since theregion of from about position 44 to about position 126 of SEQ ID NO: 36can be modified by one or more deletions in accordance with theinvention, a fragment does not have to contain the entire region. Hence,fragments shorter than the length of the unmodified region are envisagedby the invention. A fragment preferably comprises the pore formingdomain of SEQ ID NO: 36. A fragment more preferably comprises the regionfrom about position 44 to about position 126 of SEQ ID NO: 36.

One or more amino acids may be alternatively or additionally added tothe variants described above. An extension may be provided at the aminoterminal or carboxy terminal of the amino acid sequence of the variantof SEQ ID NO: 36, including a fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 36 and which retains itsability to form a pore. A variant typically contains the region of SEQID NO: 36 that is responsible for pore formation, namely from aboutposition 44 to about position 126. It may contain a fragment of thisregion as discussed above.

The variant of SEQ ID NO: 36 may be any of those disclosed inInternational Application No. PCT/GB2013/050667 (published as WO2013/153359).

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. Suitable modifications are discussed below with reference to themutant Msp monomers. Such modifications can be applied to any of thepores used in the invention.

Polynucleotide Binding Protein

Suitable polynucleotide binding proteins are discussed below. Theunmodified polynucleotide binding protein used in the inventionpreferably comprises the sequence shown in SEQ ID NO: 24 or a variantthereof. Suitable variants of SEQ ID NO: 24 are discussed below.

In SEQ ID NO: 24 or a variant thereof, the part of the polynucleotidebinding protein which interacts with the transmembrane pore typicallycomprises the amino acids at positions 1, 2, 3, 4, 5, 6, 51, 176, 177,178, 179, 180, 181, 185, 189, 191, 193, 194, 195, 197, 198, 199, 200,201, 202, 203, 204, 207, 208, 209, 210, 211, 212, 213, 216, 219, 220,221, 223, 224, 226, 227, 228, 229, 247, 254, 255, 256, 257, 258, 259,260, 261, 298, 300, 304, 308, 318, 319, 321, 337, 347, 350, 351, 405,415, 422, 434, 437, 438. These numbers correspond to the relevantpositions in SEQ ID NO: 24 and may need to be altered in the case ofvariants where one or more amino acids have been inserted or deletedcompared with SEQ ID NO: 24. A skilled person can determine thecorresponding positions in a variant as discussed above. The part of thepolynucleotide binding protein which interacts with the transmembranepore preferably comprises the amino acids at

(a) positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198,199, 200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223,224, 226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321,347, 350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24 or atthe corresponding positions in the variant thereof; or

(b) positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202,203, 207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304,350 and 438 in SEQ ID NO: 24 or at the corresponding positions in thevariant thereof.

The part of the polynucleotide binding protein which interacts with thetransmembrane pore preferably comprises one or more of, such as 2, 3 or4 of, the amino acids at positions 195, 198, 199 and 258 in SEQ ID NO:24 or the variant thereof. The part of the polynucleotide bindingprotein which interacts with the transmembrane pore preferably comprisesthe amino acid at position 195, 198, 199 or 258 in SEQ ID NO: 24 or atthe corresponding positions in the variant thereof. The modifiedpolynucleotide binding protein of the invention preferably comprises avariant of SEQ ID NO: 24 which comprises one or more of the followingmodifications (a) W195A, (b) D198V, (c) K199L or (d) E258L. The variantmay comprise {a}; {b}; {c}; {d}; {a,b}; {a,c}; {a,d}; {b,c}; {b,d};{c,d}; {a,b,c}; {a,b,d}; {a,c,d}; {b,c,d}; or {a,b,c,d}. The variant ofSEQ ID NO: 24 may further comprise any of the additional modificationsdiscussed below. The modifications set out in this paragraph arepreferred when the modified polynucleotide binding protein interactswith a pore derived from MspA, particularly any of the modified pores ofthe invention.

The part of the polynucleotide binding protein which interacts with thetransmembrane pore preferably comprises the amino acid at position 199of SEQ ID NO: 24 or at the corresponding position in the variantthereof. The modified polynucleotide binding protein of the inventionpreferably comprises a variant of SEQ ID NO: 24 which comprises K199A,K199V, K199F, K199D, K199S, K199W or K199L.

The unmodified polynucleotide binding protein used in the inventionpreferably comprises the sequence shown in SEQ ID NO: 9 or a variantthereof. A variant of SEQ ID NO: 9 is a protein that has an amino acidsequence which varies from that of SEQ ID NO: 9 and which retains itsability to bind a polynucleotide. The ability of a variant to bind apolynucleotide can be assayed using any method known in the art.

Over the entire length of the amino acid sequence of SEQ ID NO: 9, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 9 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed below.

The variant may be modified in any of the ways discussed above withreference to other variants. Preferred variants of SEQ ID NO: 9 lackamino acids 1 to 4.

In SEQ ID NO: 9 or variant thereof, the part of the polynucleotidebinding protein which interacts with the transmembrane pore typicallycomprises the amino acids at positions 80, 81, 82, 84, 85, 205, 206,209, 215, 216, 220, 221, 224, 236, 240, 241, 267, 270, 272, 278, 287,289, 293, 296, 307, 308, 309, 310, 320, 321, 322, 323, 327, 349, 415,418 and 419. These numbers correspond to the relevant positions in SEQID NO: 9 and may need to be altered in the case of variants where one ormore amino acids have been inserted or deleted. A skilled person candetermine the corresponding positions in a variant as discussed above.

The part of the polynucleotide binding protein which interacts with thetransmembrane pore preferably comprises the amino acids at

(a) positions 80, 84, 205, 209, 215, 216, 221, 224, 236, 241, 267, 272,289, 296, 307, 308, 309, 320, 321, 322, and 419 in SEQ ID NO: 9 or atthe corresponding positions in a variant thereof;

(b) positions 80, 84, 209, 215, 216, 221, 267, 272, 289, 307, 308, 309,321 and 322 in SEQ ID NO: 9 or at the corresponding positions in avariant thereof; or

(b) positions 215, 267, 272, 307, 308 and 322 in SEQ ID NO: 9 or at thecorresponding positions in a variant thereof.

Any of these positions may be replaced with phenylalanine (F),tryptophan (W), isoleucine (I), leucine (L), valine (V), alanine (A),arginine (R), lysine (K), aspartic acid (D), glutamic acid (E) ortyrosine (Y) in accordance with the invention.

Parts which Interact

The part of the transmembrane pore which may be modified in accordancewith the invention is typically the part of the transmembrane pore whichinteracts with or contacts the polynucleotide binding protein when theprotein controls the movement of the target polynucleotide with respectto the pore. The part may comprise one or more amino acids whichinteract with or contact one or more amino acids in the polynucleotidebinding protein when the protein controls the movement of the targetpolynucleotide with respect to the pore. Specific amino acids arediscussed in more detail below.

The part of the polynucleotide binding protein which may be modified inaccordance with the invention is typically the part of the protein whichinteracts with or contacts the transmembrane pore when the proteincontrols the movement of the target polynucleotide with respect to thepore. The part may comprise one or more amino acids which interact withor contact one or more amino acids in the transmembrane pore when theprotein controls the movement of the target polynucleotide with respectto the pore. Specific amino acids are discussed in more detail below.

The part of the transmembrane pore which interacts with thepolynucleotide binding protein and/or the part of the polynucleotidebinding protein which interacts with the transmembrane pore can beidentified using any method known in the art. The part(s) may beidentified using protein modelling, x-ray diffraction measurement of theprotein in a crystalline state (Rupp B (2009). BiomolecularCrystallography: Principles, Practice and Application to StructuralBiology. New York: Garland Science.), nuclear magnetic resonance (NMR)spectroscopy of the protein in solution (Mark Rance; Cavanagh, John;Wayne J. Fairbrother; Arthur W. Hunt III; Skelton, Nicholas J. (2007).Protein NMR spectroscopy: principles and practice (2nd ed.). Boston:Academic Press.) or cryo-electron microscopy of the protein in afrozen-hydrated state (van Heel M, Gowen B, Matadeen R, Orlova E V, FinnR, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000).“Single-particle electron cryo-microscopy: towards atomic resolution.” QRev Biophys. 33: 307-69. Structural information of proteins determinedby above mentioned methods are publicly available from the protein bank(PDB) database.

Protein modelling exploits the fact that protein structures are moreconserved than protein sequences amongst homologues. Hence, producingatomic resolution models of proteins is dependent upon theidentification of one or more protein structures that are likely toresemble the structure of the query sequence. In order to assess whethera suitable protein structure exists to use as a “template” to build aprotein model, a search is performed on the protein data bank (PDB)database. A protein structure is considered a suitable template if itshares a reasonable level of sequence identity with the query sequence.If such a template exists, then the template sequence is “aligned” withthe query sequence, i.e. residues in the query sequence are mapped ontothe template residues. The sequence alignment and template structure arethen used to produce a structural model of the query sequence. Hence,the quality of a protein model is dependent upon the quality of thesequence alignment and the template structure.

The part(s) may also be identified using molecular simulations or energyminimisations (Kalli A C, Campbell I D, Sansom M S P, (2013)“Conformational Changes in Talin on Binding to Anionic PhopholipidMembranes Facilitate Signaling by Integrin Transmembrane Helices” PLOSComputational Biology, 9, 10, e1003316 and Durrieu M, Lavery R, BaadenM, (2008) “Interactions between Neuronal Fusion Proteins Explored byMolecular Dynamics”, Biophysical Journal, 94, 3436-3446).

Surface

The method preferably comprises making one or modifications to thesurface of the transmembrane pore which interacts with thepolynucleotide binding protein and/or to the surface of thepolynucleotide binding protein which interacts with the transmembranepore. Any number of modifications can be made, such as 2 or more, 3 ormore, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 50 or more or 100 or more modifications.

The surface of the transmembrane pore and/or the polynucleotide bindingprotein may be identified using any method in the art, including any ofthe methods disclosed above. For instance, the surface may be identifiedusing protein modeling, molecular simulations or energy minimisations.Protein modelling is a general term to describe e.g. making models ofproteins of unknown structure, simulating proteins to investigatedynamics, molecular docking.

Pore Entrance

The method comprises making one or modifications to the entrance of thetransmembrane pore which interacts with the polynucleotide bindingprotein. Any number of modifications may be made as discussed above. Theentrance of the pore may be identified as discussed above.

Modifications

Any modifications may be made in accordance with the invention. Themethod may involve making one or more modifications which (a) alter thecharge, (b) alter the sterics, (c) alter the hydrogen bonding, (d) alterthe π stacking or (e) alter the structure of the part of thetransmembrane pore which interacts with the polynucleotide bindingprotein and/or the part of the polynucleotide binding protein whichinteracts with the transmembrane pore. Any number and combination ofthese may be altered. For instance, the method may involve making one ormore modification which {a}; {b}; {c}; {d}; {e}; {a,b}; {a,c}; {a,d};{a,e}; {b,c}; {b,d}; {b,e}; {c,d}; {c,e}; {d,e}; {a,b,c}; {a,b,d};{a,b,e}; {a,c,d}; {a,c,e}; {a,d,e}; {b,c,d}; {b,c,e}; {b,d,e}; {c,d,e};{a,b,c,d}; {a,b,c,e}; {a,b,d,e}; {a,c,d,e}; {b,c,d,e}; or {a,b,c,d,e}.

When modifying a protein, the one or more modifications typicallyinvolve introducing or replacing one or more amino acids. The inventiontypically involves making one or more amino acid substitutions.

Modifications which alter the charge may involve increasing the netnegative charge or decreasing the net negative charge. The methodpreferably comprises making one or more modifications which decrease thenet negative charge of the part of the transmembrane pore whichinteracts with the polynucleotide binding protein. Modifications whichdecrease net negative charge are discussed in more detail below withreference to mutant Msp monomers. Any of these modifications may be madein other transmembrane pores and/or polynucleotide binding proteins. Ina preferred embodiment, the transmembrane pore comprises seven or moremonomers, such as 8 or 9 monomers, comprising SEQ ID NO: 2 of a variantthereof and the method preferably comprises modifying one or more of theseven or more monomers, such as 2, 3, 4, 5, 6, 7, 8 or 9 of themonomers, so they do not comprise aspartic acid (D) or glutamic acid (E)at one or more of positions 56, 57, 59, 134 and 139 in SEQ ID NO: 2 orat one or more of the corresponding positions in the variant thereof.The method more preferably comprises modifying one or more of themonomers, such as 2, 3, 4, 5, 6, 7, 8 or 9 of the monomers, so theycomprise one or more of (a) D56N, D56R, D56F, D56Y, D56L D56K, and/or(b) E57N, E57R, E57F, E57Y, E57L or E57K, and/or (c) E59N, E59R, E59F,E59Y, E59L, E59K, and/or (d) D134N, D134R, D134F, D134Y, D134L, D134K,and/or (e) E139N, E139R, E139F, E139Y, E139L or E139K. One or more ofthe monomers may comprise any number and combination of thesemodifications. For instance, one or more of the monomers may comprise{a}; {b}; {c}; {d}; {e}; {a,b}; {a,c}; {a,d}; {a,e}; {b,c}; {b,d};{b,e}; {c,d}; {c,e}; {d,e}; {a,b,c}; {a,b,d}; {a,b,e}; {a,c,d}; {a,c,e};{a,d,e}; {b,c,d}; {b,c,e}; {b,d,e}; {c,d,e}; {a,b,c,d}; {a,b,c,e};{a,b,d,e}; {a,c,d,e}; {b,c,d,e}; or {a,b,c,d,e}. One or more of themonomers may comprise D56N and E59R, D56F and E59R, D56N and E59F, D56Nand E59Y or D56L and E59L. One or more of the monomers may comprise D56Nand E59R, D56F and E59R or D56N and E59F.

The modified transmembrane pore (i.e. the transmembrane pore resultingfrom the modification method of the invention) preferably does notcomprise one or more monomers, such as 2, 3, 4, 5, 6, 7, 8 or 9monomers, which are variants of SEQ ID NO: 2 comprising or consisting ofE59R, D90N, D91N, D93N, D118R, D134R and E139K.

Modifications which alter the sterics may involve increasing ordecreasing the size of amino acid residues, for instance bysubstitution. For instance, sterics can be increased by the introductionof one or more bulky amino acids, such as phenylalanine (F), tryptophan(W), tyrosine (Y) and histidine (H).

Modifications which alter the hydrogen bonding may involve theintroduction or replacement of one or more amino acids which canhydrogen bond.

Modifications which alter the π stacking may involve the introduction orreplacement of amino acids that interact through delocalised electron itsystems. For instance, π stacking can be increased by the introductionof one or more aromatic amino acids, such as phenylalanine (F),tryptophan (W), tyrosine (Y) and histidine (H).

In a preferred embodiment, the transmembrane pore comprises one or moremonomers comprising SEQ ID NO: 36 of a variant thereof and the methodpreferably comprises modifying one or more of the monomers so theycomprise phenylalanine (F), tryptophan (W), isoleucine (I), leucine (L),valine (V), alanine (A), arginine (R), lysine (K), aspartic acid (D),glutamic acid (E) or tyrosine (Y) at one or more of positions (i) 31,(ii) 33, (iii) 108, (iv) 109, (v) 110 and (vi) 138 in SEQ ID NO: 36 orat one or more of the corresponding positions in the variant thereof,such as {i}, {ii}, {iii}, {iv}, {v}, {vi}, {i,ii}, {i,iii}, {i,iv},{i,v}, {i,vi}, {ii,iii}, {ii,iv}, {ii,v}, {ii,vi}, {iii,iv}, {iii,v},{iii,vi}, {iv,v}, {iv,vi}, {v,vi}, {i,ii,iii}, {i,ii,iv}, {i,ii,v},{i,ii,vi}, {i,iii,iv}, {i,iii,v}, {i,iii,vi}, {i,iv,v}, {i,iv,vi},{i,v,vi}, {ii,iii,iv}, {ii,iii,v}, {ii,iii,vi}, {ii,iv,v}, {ii,iv,vi},{ii,v,vi}, {iii,iv,v}, {iii,iv,vi}, {iii,v,vi}, {iv,v,vi},{i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iii,vi}, {i,ii,iv,v}, {i,ii,iv,vi},{i,ii,v,vi}, {i,iii,iv,v}, {i,iii,iv,vi}, {i,iii,v,vi}, {i,iv,v,vi},{ii,iii,iv,v}, {ii,iii,iv,vi}, {ii,iii,v,vi}, {ii,iv,v,vi},{iii,iv,v,vi}, {i,ii,iii,iv,v}, {i,ii,iii,iv,vi}, {i,ii,iii,v,vi},{i,ii,iv,v,vi}, {i,iii,iv,v,vi}, {ii,iii,iv,v,vi} or {i,ii,iii,iv,v,vi}.

In a preferred embodiment, the transmembrane pore comprises sevenmonomers comprising SEQ ID NO: 4 of a variant thereof and the methodpreferably comprises modifying one or more of the monomers, such as 1,2, 3, 4, 5, 6 or 7 of the monomers, so they comprise phenylalanine (F),tryptophan (W), isoleucine (I), leucine (L), valine (V), alanine (A),arginine (R), lysine (K), aspartic acid (D), glutamic acid (E) ortyrosine (Y) at one or more of positions 16, 17, 18, 19, 21, 46, 47, 93,236, 237, 238, 239, 240, 241, 242, 281, 283, 285, 287, 288 and 293 inSEQ ID NO: 4 or at one or more of the corresponding positions in thevariant thereof.

In a more preferred embodiment, the transmembrane pore comprises sevenmonomers comprising SEQ ID NO: 4 of a variant thereof and the methodpreferably comprises modifying one or more of the monomers, such as 1,2, 3, 4, 5, 6 or 7 of the monomers, so they comprise phenylalanine (F),tryptophan (W), isoleucine (I), leucine (L), valine (V), alanine (A),arginine (R), lysine (K), aspartic acid (D), glutamic acid (E) ortyrosine (Y) at one or more of positions

(a) 17, 18, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242, 287, 288and 293 in SEQ ID NO: 4 or at one or more of the corresponding positionsin the variant thereof;

(b) 17, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242 and 287 in SEQID NO: 4 or at one or more of the corresponding positions in the variantthereof; or

(c) 17, 19, 46, 93, 236, 237, 239, 240, 287 and 288 in SEQ ID NO: 4 orat one or more of the corresponding positions in the variant thereof.

Modified Pores

The method also provides a transmembrane pore modified in accordancewith the invention. A part of the transmembrane pore which interactswith a polynucleotide binding protein has been modified. The part of thetransmembrane pore which interacts with a polynucleotide binding proteinwhen the polynucleotide binding protein is used to control the movementof a target polynucleotide with respect to, or through, the pore hasbeen modified. Any of the modifications discussed above may be made inthe pores of the invention.

The transmembrane pore preferably comprises seven or more monomers, suchas 8 or 9 monomers, comprising the sequence shown in SEQ ID NO: 2 or avariant thereof. One or more of the seven or more monomers, such as 2,3, 4, 5, 6, 7, 8 or 9 of the monomers, preferably comprises a variant ofSEQ ID NO: 2 which comprises one or more of (a) D56N, D56R, D56F, D56Yor D56L, (b) E57N or E57R, (c) E59N, E59R, E59F, E59Y or E59L, (d) D134Nor D134R and (e) E139N, E139R or E139K. Any number and combination ofthese modifications may be made in a single monomer as discussed above.

One or more of the seven or more monomers, such as 2, 3, 4, 5, 6, 7, 8or 9 of the monomers, preferably comprises a variant of SEQ ID NO: 2which comprises D56N, D56R, D56F, D56Y or D56L.

One or more of the seven or more monomers, such as 2, 3, 4, 5, 6, 7, 8or 9 of the monomers, preferably comprises a variant of SEQ ID NO: 2which comprises E59N, E59R, E59F, E59Y or E59L.

One or more of the seven or more monomers, such as 2, 3, 4, 5, 6, 7, 8or 9 of the monomers, preferably comprises a variant of SEQ ID NO: 2which comprises D56N and E59R, D56F and E59R, D56N and E59F, D56N andE59Y or D56L and E59L.

One or more of the seven or more monomers, such as 2, 3, 4, 5, 6, 7, 8or 9 of the monomers, preferably comprises a variant of SEQ ID NO: 2which comprises D56N and E59R, D56F and E59R or D56N and E59F.

One or more of the seven or more monomers, such as 2, 3, 4, 5, 6, 7, 8or 9 of the monomers, are not variants of SEQ ID NO: 2 which comprise orconsist of E59R, D90N, D91N, D93N, D118R, D134R and E139K.

The transmembrane pore preferably comprises one or more monomerscomprising a variant of SEQ ID NO: 36 which comprises phenylalanine (F),tryptophan (W), isoleucine (I), leucine (L), valine (V), alanine (A),arginine (R), lysine (K), aspartic acid (D), glutamic acid (E) ortyrosine (Y) at one or more of positions (i) 31, (ii) 33, (iii) 108,(iv) 109, (v) 110 and (vi) 138 or at one or more the correspondingpositions thereof, such as {i}, {ii}, {iii}, {iv}, {v}, {vi}, {i,ii},{i,iii}, {i,iv}, {i,v}, {i,vi}, {ii,iii}, {ii,iv}, {ii,v}, {ii,vi},{iii,iv}, {iii,v}, {iii,vi}, {iv,v}, {iv,vi}, {v,vi}, {i,ii,iii},{i,ii,iv}, {i,ii,v}, {i,ii,vi}, {i,iii,iv}, {i,iii,v}, {i,iii,vi},{i,iv,v}, {i,iv,vi}, {i,v,vi}, {ii,iii,iv}, {ii,iii,v}, {ii,iii,vi},{ii,iv,v}, {ii,iv,vi}, {ii,v,vi}, {iii,iv,v}, {iii,iv,vi}, {iii,v,vi},{iv,v,vi}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iii,vi}, {i,ii,iv,v},{i,ii, iv,vi}, {i,ii,v,vi}, {i,iii,iv,v}, {i,iii,iv,vi}, {i,iii,v,vi},{i,iv,v,vi}, {ii,iii,iv,v}, {ii,iii,iv,vi}, {ii,iii,v,vi}, {ii,iv,v,vi},{iii,iv,v,vi}, {i,ii,iii,iv,v}, {i,ii,iii,iv,vi}, {i,ii,iii,v,vi},{i,ii,iv,v,vi}, {i,iii,iv,v,vi}, {ii,iii,iv,v,vi} or {i,ii,iii,iv,v,vi}.

The transmembrane pore preferably comprises seven monomers comprisingSEQ ID NO: 4 or a variant thereof in which one or more of the monomers,such as 1, 2, 3, 4, 5, 6 or 7 of the monomers, is a variant of SEQ IDNO: 4 which comprises phenylalanine (F), tryptophan (W), isoleucine (I),leucine (L), valine (V), alanine (A), arginine (R), lysine (K), asparticacid (D), glutamic acid (E) or tyrosine (Y) at one or more of positions16, 17, 18, 19, 21, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242, 281,283, 285, 287, 288 and 293 or at one or more of the correspondingpositions thereof, such as one or more of positions:

(a) 17, 18, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242, 287, 288and 293 or at one or more of the corresponding positions thereof;

(b) 17, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242 and 287 in SEQID NO: 4 or at one or more of the corresponding positions thereof; or

(c) 17, 19, 46, 93, 236, 237, 239, 240, 287 and 288 in SEQ ID NO: 4 orat one or more of the corresponding positions thereof.

Mutant Msp Monomers

The present invention also provides mutant Msp monomers. The mutant Mspmonomers may be used to form the pores of the invention. A mutant Mspmonomer is a monomer whose sequence varies from that of a wild-type Mspmonomer and which retains the ability to form a pore. Methods forconfirming the ability of mutant monomers to form pores are well-knownin the art and are discussed in more detail below.

The mutant monomers have improved polynucleotide reading properties i.e.display improved polynucleotide capture and nucleotide discrimination.In particular, pores constructed from the mutant monomers capturenucleotides and polynucleotides more easily than the wild type. Inaddition, pores constructed from the mutant monomers display anincreased current range, which makes it easier to discriminate betweendifferent nucleotides, and a reduced variance of states, which increasesthe signal-to-noise ratio. In addition, the number of nucleotidescontributing to the current as the polynucleotide moves with respect to,or through, pores constructed from the mutants is decreased. This makesit easier to identify a direct relationship between the observed currentas the polynucleotide moves with respect to, or through, the pore andthe polynucleotide sequence.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 2. SEQ ID NO: 2 is the wild-type MspA monomer. Avariant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequencewhich varies from that of SEQ ID NO: 2 and which retains its ability toform a pore. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into an amphiphilic layer along with other appropriate subunitsand its ability to oligomerise to form a pore may be determined. Methodsare known in the art for inserting subunits into membranes, such asamphiphilic layers. For example, subunits may be suspended in a purifiedform in a solution containing a triblock copolymer membrane such that itdiffuses to the membrane and is inserted by binding to the membrane andassembling into a functional state.

The mutant Msp monomer is modified in accordance with the invention. Apart of the monomer which interacts with a polynucleotide bindingprotein has been modified. The part of the monomer which interacts witha polynucleotide binding protein when the polynucleotide binding proteinis used to control the movement of a target polynucleotide with respectto, or through, a pore comprising the monomer has been modified. Any ofthe modifications discussed above may be made in the monomer of theinvention.

The monomer preferably comprises a variant of SEQ ID NO: 2 whichcomprises one or more of (a) D56N, D56R, D56F, D56Y or D56L, (b) E57N orE57R, (c) E59N, E59R, E59F, E59Y or E59L, (d) D134N or D134R and (e)E139N, E139R or E139K. Any number and combination of these modificationsmay be made in the monomer as discussed above. The variant preferablycomprises D56N and E59R, D56F and E59R, D56N and E59F, D56N and E59Y orD56L and E59L. The variant preferably comprises D56N and E59R, D56F andE59R or D56N and E59F. The monomer preferably does not comprise avariant of SEQ ID NO: 2 which comprises or consists of E59R, D90N, D91N,D93N, D118R, D134R and E139K.

Barrel Deletions

In the variant (of SEQ ID NO: 2), 2, 4, 6, 8 or 10 of the amino acids atpositions 72 to 82 of SEQ ID NO: 2 may have been deleted. 2, 4, 6, 8 or10 of the amino acids at positions 111 to 121 of SEQ ID NO: 2 may havealso been deleted. In other words, 2, 4, 6, 8 or 10 amino acids may bedeleted from the downward strand (positions 72 to 82) and the upwardstrand (positions 111 to 121) of the barrel region of SEQ ID NO: 2.These deletions and their advantages are discussed in more detail in UKApplication No. 1417708.3 co-filed with this application.

The number of amino acids deleted from positions 72 to 82 may bedifferent from the number of amino acids deleted from positions 111 to121. The number of amino acids deleted from positions 72 to 82 ispreferably the same as the number of amino acids deleted from positions111 to 121.

Any combination of amino acids from positions 72 to 82 and amino acidsfrom positions 111 to 121 may be deleted. The majority of the aminoacids in the downward and upwards strands of the barrel in SEQ ID NO: 2alternate between hydrophobic and hydrophilic. The hydrophobic aminoacids are selected from tryptophan (W), leucine (L), valine (V),isoleucine (I), phenylalanine (F) and tyrosine (Y). The hydrophilicamino acids are selected from serine (S), glycine (G), asparagine (N),proline (P) and aspartic acid (D).

Positions 72 to 82 of SEQ ID NO: 2 correspond to W-S-L-G-V-G-I-N-F-S-Y(SEQ ID NO: 26 with the hydrophobic amino acids underlined). Positions111 to 121 of SEQ ID NO: 2 correspond to P-G-V-S-I-S-A-D-L-G-N(SEQ IDNO: 27 with the hydrophobic amino acids underlined). This alternationbetween hydrophobic and hydrophilic amino acids results in thebeta-sheet which forms part of the barrel of the pore.

The amino acids from positions 72 to 82 remaining after deletion (i.e.after 2, 4, 6, 8 or 10 amino acids have been deleted from positions 72to 82) preferably comprise 3, 5, 7 or 9 consecutive amino acids whichalternate between hydrophobic and hydrophilic.

The amino acids from positions 111 to 121 remaining after deletion (i.e.after 2, 4, 6, 8 or 10 amino acids have been deleted from positions 111to 121) preferably comprise 3, 5, 7 or 9 consecutive amino acids whichalternate between hydrophobic and hydrophilic.

The amino acids deleted from positions 72 to 82 may correspond to theamino acids deleted from positions 111 to 121 as shown in Table 1 below.For instance, if L74 and G75 are deleted from positions 72 to 82, D118and L119 may be deleted from positions 111 to 121.

TABLE 1 Corresponding amino acids in the barrel of SEQ ID NO: 2 Positionin (a) Corresponding position in (b) W72 N121 S73 G120 L74 L119 G75 D118V76 A117 G77 S116 I78 I115 N79 S114 F80 V113 S81 G112 Y82 P111

One or more positions of the amino acids that have been deleted frompositions 72 to 82 may not correspond to the one or more positions ofthe amino acids that have been deleted from positions 111 to 121 asshown in Table 1. For instance, if L74 and G75 are deleted frompositions 72 to 82, A117 and D118 may be deleted from positions 111 to121.

The positions of (all of) the amino acids that have been deleted frompositions 72 to 82 may not correspond to the positions of (all of) theamino acids that have been deleted from positions 111 to 121 as shown inTable 1. For instance, if L74 and G75 are deleted from positions 72 to82, I115 and S116 may be deleted from positions 111 to 121.

The amino acids deleted from positions 72 to 82 are preferablyconsecutive. The amino acids deleted from positions 111 to 121 arepreferably consecutive. The amino acids deleted from positions 72 to 82and the amino acids deleted from positions 111 to 121 are preferablyconsecutive.

The invention preferably provides mutant monomers comprising a variantof the sequence shown in SEQ ID NO: 2, wherein in the variant (i) L74,G75, D118 and L119 have been deleted, (ii) G75, V76, A117 and D118 havebeen deleted, (iii) V76, G77, S116 and A117 have been deleted, (iv) G77,178, 1115 and S116 have been deleted, (v) 178, N79, S114 and 1115 havebeen deleted, (vi) N79, F80, V113 and S114 have been deleted or (vii)F80, S81, G112 and V113 have been deleted. The invention preferablycomprises a variant of the sequence shown in SEQ ID NO: 2, wherein inthe variant L74, G75, V76, G77, S116, A117, D118 and L119 have beendeleted. The invention preferably comprises a variant of the sequenceshown in SEQ ID NO: 2, wherein in the variant L74, G75, N79, F80, V113,S114, D118 and L119 or L74, G75, F80, S81, G112, V113, D118 and L119.

The skilled person can identify other combinations of amino acids thatmay be deleted in accordance with the invention. The followingdiscussion using the numbering of residues in SEQ ID NO: 2 (i.e. beforeany amino acids have been deleted as defined above).

Positions 90 and 91

In wild-type MspA, amino acids 90 and 91 are both aspartic acid (D).These amino acids in each monomer form part of an inner constriction ofthe pore. The variant preferably does not comprise aspartic acid (D) atposition 90. The variant preferably does not comprise aspartic acid (D)or glutamic acid (E) at position 90. The variant preferably does nothave a negatively charged amino acid at position 90.

The variant preferably does not comprise aspartic acid (D) at position91. The variant preferably does not comprise aspartic acid (D) orglutamic acid (E) at position 91. The variant preferably does not have anegatively charged amino acid at position 91.

The variant preferably comprises serine (S), glutamine (Q), leucine (L),methionine (M), isoleucine (I), alanine (A), valine (V), glycine (G),phenylalanine (F), tryptophan (W), tyrosine (Y), histidine (H),threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C)at position 90 and/or position 91. Any combinations of these amino acidsat positions 90 and 91 are envisaged by the invention. The variantpreferably comprises asparagine (N) at position 90 and/or position 91.The variant more preferably comprises asparagine (N) at position 90 andposition 91. These amino acids are preferably inserted at position 90and/or 91 by substitution.

Position 93

In wild-type MspA, amino acid 93 is aspartic acid (D). This amino acidin each monomer also forms part of an inner constriction of the pore.

The variant preferably comprises aspartic acid (D) or glutamic acid (E)at position 93. The variant preferably has a negative charge at position93. The glutamic acid (E) is preferably introduced by substitution.

Cap Forming Region

In wild-type MspA, amino acids 1 to 72 and 122 to 184 form the cap ofthe pore. Of these amino acids, V9, Q12, D13, R14, T15, W40, 149, P53,G54, D56, E57, E59, T61, E63, Y66, Q67, I68, F70, P123, I125, Q126,E127, V128, A129, T130, F131, S132, V133, D134, S136, G137, E139, V144,H148, T150, V151, T152, F163, R165, 1167, S169, T170 and S173 faceinwards into the channel of the pore.

Barrel Forming Region

In wild-type MspA, amino acids 72 to 82 and 112 to 121 form the barrelof the pore. Of these amino acids, S73, G75, G77, N79, S81, G112, S114,S116, D118 and G120 face inwards into the channel of the pore. S73, G75,G77, N79, S81 face inwards in the downwards strand and G112, S114, S116,D118 and G120 face inwards in the upwards strand.

Decreased Net Negative Charge

The variant preferably comprises one or more modifications whichdecrease the net negative charge of the inward facing amino acids in thecap forming region and/or the barrel forming region of the monomer. Thevariant preferably comprises two or more modifications which decreasethe net negative charge of the inward facing amino acids in the capforming region and the barrel forming region of the monomer. Any suchmodifications to the barrel forming region are in addition to thedeletions of the invention discussed above.

The variant may comprise any number of modifications, such as 1 or more,2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40or more modifications.

The net negative charge may be decreased by any means known in the art.The net negative charge is decreased in a manner that does not interferewith the ability of the mutant monomer to form a pore. This can bemeasured as discussed above.

The net negative charge of the inward facing amino acids in the capforming region and/or the barrel forming region may be decreased. Thismeans that the inward facing amino acids in the cap forming regionand/or the barrel forming region comprise fewer negatively charged aminoacids than in SEQ ID NO: 2 and/or comprises more positively chargedamino acids than in SEQ ID NO: 2. The one or more modifications may leadto a net positive charge in the inward facing amino acids in the capforming region and/or the barrel forming region

The net charge can be measured using methods known in the art. Forinstance, the isolectric point may be used to define the net charge ofthe inward facing amino acids in the cap forming region and/or thebarrel forming region.

The one or more modifications are preferably one or more deletions ofnegatively charged amino acids. Removal of one or more negativelycharged amino acids reduces the net negative charge of the inward facingamino acids in the cap forming region and/or barrel forming region. Anegatively charged amino acid is an amino acid with a net negativecharge. Negatively charged amino acids include, but are not limited to,aspartic acid (D) and glutamic acid (E). Methods for deleting aminoacids from proteins, such as MspA monomers, are well known in the art.

The one or more modifications are preferably one or more substitutionsof negatively charged amino acids with one or more positively charged,uncharged, non-polar and/or aromatic amino acids. A positively chargedamino acid is an amino acid with a net positive charge. The positivelycharged amino acid(s) can be naturally-occurring ornon-naturally-occurring. The positively charged amino acid(s) may besynthetic or modified. For instance, modified amino acids with a netpositive charge may be specifically designed for use in the invention. Anumber of different types of modification to amino acids are well knownin the art.

Preferred naturally-occurring positively charged amino acids include,but are not limited to, histidine (H), lysine (K) and arginine (R). Anynumber and combination of H, K and/or R may be substituted for theinward facing amino acids in the cap forming region and/or barrelforming region.

The uncharged amino acids, non-polar amino acids and/or aromatic aminoacids can be naturally occurring or non-naturally-occurring. They may besynthetic or modified. Uncharged amino acids have no net charge.Suitable uncharged amino acids include, but are not limited to, cysteine(C), serine (S), threonine (T), methionine (M), asparagines (N) andglutamine (Q). Non-polar amino acids have non-polar side chains.Suitable non-polar amino acids include, but are not limited to, glycine(G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine(V). Aromatic amino acids have an aromatic side chain. Suitable aromaticamino acids include, but are not limited to, histidine (H),phenylalanine (F), tryptophan (W) and tyrosine (Y). Any number andcombination of these amino acids may be substituted into the inwardfacing amino acids in the cap forming region and/or the barrel formingregion.

The one or more negatively charged amino acids are preferablysubstituted with alanine (A), valine (V), asparagine (N) or glycine (G).Preferred substitutions include, but are not limited to, substitution ofD with A, substitution of D with V, substitution of D with N andsubstitution of D with G.

The one or more modifications are preferably one or more introductionsof positively charged amino acids. The introduction of positive chargedecreases the net negative charge. The one or more positively chargedamino acids may be introduced by addition or substitution. Any aminoacid may be substituted with a positively charged amino acid. One ormore uncharged amino acids, non-polar amino acids and/or aromatic aminoacids may be substituted with one or more positively charged aminoacids. Any number of positively charged amino acids may be introduced.

Wild-type MspA comprises a polar glutamine (Q) at position 126. The oneor more modifications preferably reduce the net negative charge atposition 126. The one or more modifications preferably increase the netpositive charge at positions 126. This can be achieved by replacing thepolar amino acid at position 126 or an adjacent or a nearby inwardfacing amino acid with a positively charged amino acid. The variantpreferably comprises a positively charged amino acid at position 126.The variant preferably comprises a positively charged amino acid at oneor more of positions 123, 125, 127 and 128. The variant may comprise anynumber and combination of positively charged amino acids at positions123, 125, 127 and 128. The positively charged amino acid(s) may beintroduced by addition or substitution.

The one or more modifications are preferably one or more introductionsof positively charged amino acids which neutralise one or morenegatively charged amino acids. The neutralisation of negative chargedecreases the net negative charge. The one or more positively chargedamino acids may be introduced by addition or substitution. Any aminoacid may be substituted with a positively charged amino acid. One ormore uncharged amino acids, non-polar amino acids and/or aromatic aminoacids may be substituted with one or more positively charged aminoacids. Any number of positively charged amino acids may be introduced.The number is typically the same as the number of negatively chargedamino acids being neutralised.

The one or more positively charged amino acids may be introduced at anyposition in the cap forming region and/or the barrel forming region aslong as they neutralise the negative charge of the one or more inwardfacing negatively charged amino acids. To effectively neutralise thenegative charge in the cap forming region, there is typically 5 or feweramino acids in the variant between each positively charged amino acidthat is introduced and the negatively charged amino acid it isneutralising. There are preferably 4 or fewer, 3 or fewer or 2 or feweramino acids in the cap forming region of the variant between eachpositively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. There is more preferably twoamino acids in the cap forming region of the variant between eachpositively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. Each positively charged aminoacid is most preferably introduced adjacent in the cap forming region ofthe variant to the negatively charged amino acid it is neutralising.

To effectively neutralise the negative charge in the barrel formingregion, there is typically 5 or fewer inward facing amino acids betweeneach positively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. There is preferably 4 or fewer, 3or fewer or 2 or fewer inward facing amino acids in the barrel formingregion of the variant between each positively charged amino acid that isintroduced and the negatively charged amino acid it is neutralising.There is more preferably one inward facing amino acid in the barrelforming region of the variant between each positively charged amino acidthat is introduced and the negatively charged amino acid it isneutralising. Each positively charged amino acid is most preferablyintroduced at the inward facing position adjacent in the barrel formingregion of the variant to the negatively charged amino acid it isneutralising.

Wild-type MspA comprises aspartic acid (D) at positions 118 and 134 andglutamic acid (E) at position 139. Amino acid 118 in each monomer ispresent within the barrel of the pore (FIG. 37). The variant preferablycomprises a positively charged amino acid at one or more of positions114, 116, 120, 123, 70, 73, 75, 77 and 79. Positive charges at one ormore of these positions neutralise the negative charge at position 118.Positively charged amino acids may present at any number and combinationof positions 114, 116, 120, 123, 70, 73, 75, 77 and 79. The amino acidsmay be introduced by addition or substitution.

Amino acids 134 and 139 in each monomer are part of the cap. The variantcomprises a positively charged amino acid at one or more of positions129, 132, 136, 137, 59, 61 and 63. Positive charges at one or more ofthese positions neutralise the negative charge at position 134.Positively charged amino acids may be present at any number andcombination of positions 129, 132, 136, 137, 59, 61 and 63. The aminoacids may be introduced by addition or substitution.

The variant preferably comprises a positively charged amino acid at oneor more of positions 137, 138, 141, 143, 45, 47, 49 and 51. Positivecharges at one or more of these positions neutralise the negative chargeat position 139. Positively charged amino acids may be present at anynumber and combination of positions 137, 138, 141, 143, 45, 47, 49 and51. The amino acids may be introduced by addition or substitution.

Positions 118, 126, 134 and 139

The one or more modifications preferably reduce the net negative chargeat one or more of positions 118, 126, 134 and 139. The one or moremodifications preferably reduce the net negative charge at 118; 126;134; 139; 118 and 126; 118 and 134; 118 and 139; 126 and 134; 126 and139; 134 and 139; 118, 126 and 134; 118, 126 and 139; 118, 134 and 139;126, 134 and 139; or 118, 126, 134 and 139.

The variant preferably does not comprise aspartic acid (D) or glutamicacid (E) at one or more of positions 118, 126, 134 and 139. The variantpreferably does not comprise aspartic acid (D) or glutamic acid (E) atany of the combination of positions 118, 126, 134 and 139 disclosedabove. The variant more preferably comprises arginine (R), glycine (G)or asparagine (N) at one or more of positions 118, 126, 134 and 139,such as any of the combinations of positions 118, 126, 134 and 139disclosed above. The variant most preferably comprises D118R, Q126R,D134R and E139K.

Methods for introducing or substituting naturally-occurring amino acidsare well known in the art. For instance, methionine (M) may besubstituted with arginine (R) by replacing the codon for methionine(ATG) with a codon for arginine (CGT) at the relevant position in apolynucleotide encoding the mutant monomer. The polynucleotide can thenbe expressed as discussed below.

Methods for introducing or substituting non-naturally-occurring aminoacids are also well known in the art. For instance,non-naturally-occurring amino acids may be introduced by includingsynthetic aminoacyl-tRNAs in the IVTT system used to express the mutantmonomer. Alternatively, they may be introduced by expressing the mutantmonomer in E. coli that are auxotrophic for specific amino acids in thepresence of synthetic (i.e. non-naturally-occurring) analogues of thosespecific amino acids. They may also be produced by naked ligation if themutant monomer is produced using partial peptide synthesis.

The one or more modifications are preferably one or more chemicalmodifications of one or more negatively charged amino acids whichneutralise their negative charge. For instance, the one or morenegatively charged amino acids may be reacted with a carbodiimide.

Other Modifications

The variant preferably comprises one or more of:

(a) serine (S) at position 75;

(b) serine (S) at position 77; and

(c) asparagine (N) or lysine (K) at position 88.

The variant may comprise any number and combination of (a) to (c),including (a), (b), (c), (a) and (b), (b) and (c), (a) and (c) and (a),(b) and (c). The variant preferably comprises G75S, G77S and L88N.

The variant most preferably comprises (a) D90N, D91N, D93N, D118R, D134Rand E139K, (b) L88N, D90N, D91N, D93N, D118R, D134R and E139K, (c) G75S,G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134R and E139K or (d) G75S,G77S, L88N, D90N, D91N, D118R, Q126R, D134R and E139K.

The variant preferably further comprises one or more of:

(e) phenylalanine (F) at position 89;

(f) glutamic acid (E) at position 95 and lysine (K) at position 98;

(g) aspartic acid (D) at position 96;

(h) glycine (G) at position 102;

(i) alanine (A) at position 103; and

(j) alanine (A), serine (S) or proline (P) at position 108.

The may comprise any number and combination of (e) to (j).

Variants

In addition to the specific mutations discussed above, the variant ofSEQ ID NO: 2 may include other mutations. Over the entire length of theamino acid sequence of SEQ ID NO: 2, a variant will preferably be atleast 50% homologous to that sequence based on amino acid identity. Morepreferably, the variant may be at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 100 or more, for example 125,150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the mature form of the wild-type MspA monomer. Thevariant may comprise any of the mutations in the MspB, C or D monomerscompared with MspA. The mature forms of MspB, C and D are shown in SEQID NOs: 5 to 7. In particular, the variant may comprise the followingsubstitution present in MspB: A138P. The variant may comprise one ormore of the following substitutions present in MspC: A96G, N102E andA138P. The variant may comprise one or more of the following mutationspresent in MspD: Deletion of G1, L2V, E5Q, LSV, D13G, W21A, D22E, K47T,149H, I68V, D91G, A96Q, N102D, S103T, V104I, S136K and G141A. Thevariant may comprise combinations of one or more of the mutations andsubstitutions from Msp B, C and D.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 2below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 3.

TABLE 2 Chemical properties of amino acids Ala aliphatic, hydrophobic,Met hydrophobic, neutral neutral Cys polar, hydrophobic, Asn polar,hydrophilic, neutral neutral Asp polar, hydrophilic, Pro hydrophobic,charged (−) neutral Glu polar, hydrophilic, Gln polar, hydrophilic,charged (−) neutral Phe aromatic, hydrophobic, Arg polar, hydrophilic,neutral charged (+) Gly aliphatic, Scr polar, hydrophilic, neutralneutral His aromatic, polar, hydro- Thr polar, hydrophilic, philic,charged (+) neutral Ile aliphatic, hydrophobic, Val aliphatichydrophobic, neutral neutral Lys polar, hydrophilic, Trp aromatic,hydrophobic, charged(+) neutral Leu aliphatic, hydrophobic, Tyraromatic, polar, neutral hydrophobic

TABLE 3 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ma 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of astreptavidin tag or by the addition of a signal sequence to promotetheir secretion from a cell where the monomer does not naturally containsuch a sequence. Other suitable tags are discussed in more detail below.The monomer may be labelled with a revealing label. The revealing labelmay be any suitable label which allows the monomer to be detected.Suitable labels are described below.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the monomer may be synthesized byin vitro transcription and translation (IVTT). Alternatively the monomermay be synthesized by recombinant protein expression in E. coli.Suitable methods for producing pores and monomers are discussed inInternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603). Methods for insertingpores into membranes are discussed.

In some embodiments, the mutant monomer is chemically modified. Themutant monomer can be chemically modified in any way and at any site.The mutant monomer is preferably chemically modified by attachment of amolecule to one or more cysteines (cysteine linkage), attachment of amolecule to one or more lysines, attachment of a molecule to one or morenon-natural amino acids, enzyme modification of an epitope ormodification of a terminus. Suitable methods for carrying out suchmodifications are well-known in the art. The mutant monomer may bechemically modified by the attachment of any molecule. For instance, themutant monomer may be chemically modified by attachment of a dye or afluorophore.

In some embodiments, the mutant monomer is chemically modified with amolecular adaptor that facilitates the interaction between a porecomprising the monomer and a target nucleotide or target polynucleotidesequence. The presence of the adaptor improves the host-guest chemistryof the pore and the nucleotide or polynucleotide sequence and therebyimproves the sequencing ability of pores formed from the mutant monomer.The principles of host-guest chemistry are well-known in the art. Theadaptor has an effect on the physical or chemical properties of the porethat improves its interaction with the nucleotide or polynucleotidesequence. The adaptor may alter the charge of the barrel or channel ofthe pore or specifically interact with or bind to the nucleotide orpolynucleotide sequence thereby facilitating its interaction with thepore.

The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, aspecies that is capable of hybridization, a DNA binder or interchelator,a peptide or peptide analogue, a synthetic polymer, an aromatic planarmolecule, a small positively-charged molecule or a small moleculecapable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the samesymmetry as the pore. The adaptor preferably has eight-fold symmetrysince Msp typically has eight subunits around a central axis. This isdiscussed in more detail below.

The adaptor typically interacts with the nucleotide or polynucleotidesequence via host-guest chemistry. The adaptor is typically capable ofinteracting with the nucleotide or polynucleotide sequence. The adaptorcomprises one or more chemical groups that are capable of interactingwith the nucleotide or polynucleotide sequence. The one or more chemicalgroups preferably interact with the nucleotide or polynucleotidesequence by non-covalent interactions, such as hydrophobic interactions,hydrogen bonding, Van der Waal's forces, π-cation interactions and/orelectrostatic forces. The one or more chemical groups that are capableof interacting with the nucleotide or polynucleotide sequence arepreferably positively charged. The one or more chemical groups that arecapable of interacting with the nucleotide or polynucleotide sequencemore preferably comprise amino groups. The amino groups can be attachedto primary, secondary or tertiary carbon atoms. The adaptor even morepreferably comprises a ring of amino groups, such as a ring of 6, 7 or 8amino groups. The adaptor most preferably comprises a ring of eightamino groups. A ring of protonated amino groups may interact withnegatively charged phosphate groups in the nucleotide or polynucleotidesequence.

The correct positioning of the adaptor within the pore can befacilitated by host-guest chemistry between the adaptor and the porecomprising the mutant monomer. The adaptor preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore. The adaptor more preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore via non-covalent interactions, such ashydrophobic interactions, hydrogen bonding, Van der Waal's forces,π-cation interactions and/or electrostatic forces. The chemical groupsthat are capable of interacting with one or more amino acids in the poreare typically hydroxyls or amines. The hydroxyl groups can be attachedto primary, secondary or tertiary carbon atoms. The hydroxyl groups mayform hydrogen bonds with uncharged amino acids in the pore. Any adaptorthat facilitates the interaction between the pore and the nucleotide orpolynucleotide sequence can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclicpeptides and cucurbiturils. The adaptor is preferably a cyclodextrin ora derivative thereof. The cyclodextrin or derivative thereof may be anyof those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The adaptor is more preferablyheptakis-6-amino-3-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). The guanidinogroup in gu₇-βCD has a much higher pKa than the primary amines inam₇-βCD and so is more positively charged. This gu₇-βCD adaptor may beused to increase the dwell time of the nucleotide in the pore, toincrease the accuracy of the residual current measured, as well as toincrease the base detection rate at high temperatures or low dataacquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker isused as discussed in more detail below, the adaptor is preferablyheptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin(am₆amPDP₁-βCD).

More suitable adaptors include γ-cyclodextrins, which comprise 8 sugarunits (and therefore have eight-fold symmetry). The γ-cyclodextrin maycontain a linker molecule or may be modified to comprise all or more ofthe modified sugar units used in the β-cyclodextrin examples discussedabove.

The molecular adaptor is preferably covalently attached to the mutantmonomer. The adaptor can be covalently attached to the pore using anymethod known in the art. The adaptor is typically attached via chemicallinkage. If the molecular adaptor is attached via cysteine linkage, theone or more cysteines have preferably been introduced to the mutant bysubstitution. The mutant monomers of the invention can of coursecomprise a cysteine residue at one or more of positions 88, 90, 91, 103and 105. The mutant monomer may be chemically modified by attachment ofa molecular adaptor to one or more, such as 2, 3, 4 or 5, of thesecysteines. Alternatively, the mutant monomer may be chemically modifiedby attachment of a molecule to one or more cysteines introduced at otherpositions. The molecular adaptor is preferably attached to one or moreof positions 90, 91 and 103 of SEQ ID NO: 2.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the mutant monomer before a linker is attached. The moleculemay be attached directly to the mutant monomer. The molecule ispreferably attached to the mutant monomer using a linker, such as achemical crosslinker or a peptide linker.

Suitable chemical crosslinkers are well-known in the art. Preferredcrosslinkers include 2,5-dioxopyrrolidin-1-yl3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl8-(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker issuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, themolecule is covalently attached to the bifunctional crosslinker beforethe molecule/crosslinker complex is covalently attached to the mutantmonomer but it is also possible to covalently attach the bifunctionalcrosslinker to the monomer before the bifunctional crosslinker/monomercomplex is attached to the molecule.

The linker is preferably resistant to dithiothreitol (DTT). Suitablelinkers include, but are not limited to, iodoacetamide-based andMaleimide-based linkers.

In other embodiment, the monomer may be attached to a polynucleotidebinding protein. This forms a modular sequencing system that may be usedin the methods of sequencing of the invention. Polynucleotide bindingproteins are discussed below.

The polynucleotide binding protein is preferably covalently attached tothe mutant monomer. The protein can be covalently attached to themonomer using any method known in the art. The monomer and protein maybe chemically fused or genetically fused. The monomer and protein aregenetically fused if the whole construct is expressed from a singlepolynucleotide sequence. Genetic fusion of a monomer to a polynucleotidebinding protein is discussed in International Application No.PCT/GB09/001679 (published as WO 2010/004265).

If the polynucleotide binding protein is attached via cysteine linkage,the one or more cysteines have preferably been introduced to the mutantby substitution. The mutant monomers of the invention can of coursecomprise cysteine residues at one or more of positions 10 to 15, 51 to60, 136 to 139 and 168 to 172. These positions are present in loopregions which have low conservation amongst homologues indicating thatmutations or insertions may be tolerated. They are therefore suitablefor attaching a polynucleotide binding protein. The reactivity ofcysteine residues may be enhanced by modification as described above.

The polynucleotide binding protein may be attached directly to themutant monomer or via one or more linkers. The molecule may be attachedto the mutant monomer using the hybridization linkers described inInternational Application No. PCT/GB10/000132 (published as WO2010/086602). Alternatively, peptide linkers may be used. Peptidelinkers are amino acid sequences. The length, flexibility andhydrophilicity of the peptide linker are typically designed such that itdoes not to disturb the functions of the monomer and molecule. Preferredflexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10or 16, serine and/or glycine amino acids. More preferred flexiblelinkers include (SG)₁, (SG)₂, (SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S isserine and G is glycine. Preferred rigid linkers are stretches of 2 to30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigidlinkers include (P)₁₂ wherein P is proline.

The mutant monomer may be chemically modified with a molecular adaptorand a polynucleotide binding protein.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the monomer before a linker is attached.

The molecule (with which the monomer is chemically modified) may beattached directly to the monomer or attached via a linker as disclosedin International Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be modified to assist their identificationor purification, for example by the addition of histidine residues (ahis tag), aspartic acid residues (an asp tag), a streptavidin tag, aflag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of asignal sequence to promote their secretion from a cell where thepolypeptide does not naturally contain such a sequence. An alternativeto introducing a genetic tag is to chemically react a tag onto a nativeor engineered position on the protein. An example of this would be toreact a gel-shift reagent to a cysteine engineered on the outside of theprotein. This has been demonstrated as a method for separating hemolysinhetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be labelled with a revealing label. Therevealing label may be any suitable label which allows the protein to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the monomers or pores ofthe invention, may be made synthetically or by recombinant means. Forexample, the protein may be synthesized by in vitro translation andtranscription (IVTT). The amino acid sequence of the protein may bemodified to include non-naturally occurring amino acids or to increasethe stability of the protein. When a protein is produced by syntheticmeans, such amino acids may be introduced during production. The proteinmay also be altered following either synthetic or recombinantproduction.

Proteins may also be produced using D-amino acids. For instance, theprotein may comprise a mixture of L-amino acids and D-amino acids. Thisis conventional in the art for producing such proteins or peptides.

The protein may also contain other non-specific modifications as long asthey do not interfere with the function of the protein. A number ofnon-specific side chain modifications are known in the art and may bemade to the side chains of the protein(s). Such modifications include,for example, reductive alkylation of amino acids by reaction with analdehyde followed by reduction with NaBH₄, amidination withmethylacetimidate or acylation with acetic anhydride.

Any of the proteins described herein, including the monomers and poresof the invention, can be produced using standard methods known in theart. Polynucleotide sequences encoding a protein may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a protein may be expressed in a bacterial host cell usingstandard techniques in the art. The protein may be produced in a cell byin situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

Proteins may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Constructs

The invention also provides a construct comprising two or morecovalently attached MspA monomers, wherein at least one of the monomersis a mutant monomer of the invention. The construct of the inventionretains its ability to form a pore. This may be determined as discussedabove. One or more constructs of the invention may be used to form poresfor characterising, such as sequencing, polynucleotides. The constructmay comprise at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9 or at least 10 monomers. Theconstruct preferably comprises two monomers. The two or more monomersmay be the same or different.

At least one monomer in the construct is a mutant monomer of theinvention. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more or 10 or more monomers in the construct maybe mutant monomers of the invention. All of the monomers in theconstruct are preferably mutant monomers of the invention. The mutantmonomers may be the same or different. In a preferred embodiment, theconstruct comprises two mutant monomers of the invention.

The mutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length.

The construct may comprise one or more monomers which are not mutantmonomers of the invention. MspA mutant monomers which are non mutantmonomers of the invention include monomers comprising SEQ ID NO: 2 or acomparative variant of SEQ ID NO: 2. At least one monomer in theconstruct may comprise SEQ ID NO: 2 or a comparative variant of thesequence shown in SEQ ID NO: 2 which comprises one or more of,preferably all of, D90N, D91N, D93N, D118R, D134R and E139K. At leastone monomer in the construct may be any of the monomers disclosed inInternational Application No. PCT/GB2012/050301 (published asWO/2012/107778), including those comprising a comparative variant of thesequence shown in SEQ ID NO: 2 which comprises G75S, G77S, L88N, D90N,D91N, D93N, D118R, Q126R, D134R and E139K. A comparative variant of SEQID NO: 2 is at least 50% homologous to SEQ ID NO: 2 over its entiresequence based on amino acid identity. More preferably, the comparativevariant may be at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence.

The monomers in the construct are preferably genetically fused. Monomersare genetically fused if the whole construct is expressed from a singlepolynucleotide sequence. The coding sequences of the monomers may becombined in any way to form a single polynucleotide sequence encodingthe construct.

The monomers may be genetically fused in any configuration. The monomersmay be fused via their terminal amino acids. For instance, the aminoterminus of the one monomer may be fused to the carboxy terminus ofanother monomer. The second and subsequent monomers in the construct (inthe amino to carboxy direction) may comprise a methionine at their aminoterminal ends (each of which is fused to the carboxy terminus of theprevious monomer). For instance, if M is a monomer (without an aminoterminal methionine) and mM is a monomer with an amino terminalmethionine, the construct may comprise the sequence M-mM, M-mM-mM orM-mM-mM-mM. The presences of these methionines typically results fromthe expression of the start codons (i.e. ATGs) at the 5′ end of thepolynucleotides encoding the second or subsequent monomers within thepolynucleotide encoding entire construct. The first monomer in theconstruct (in the amino to carboxy direction) may also comprise amethionine (e.g. mM-mM, mM-mM-mM or mM-mM-mM-mM).

The two or more monomers may be genetically fused directly together. Themonomers are preferably genetically fused using a linker. The linker maybe designed to constrain the mobility of the monomers. Preferred linkersare amino acid sequences (i.e. peptide linkers). Any of the peptidelinkers discussed above may be used.

In another preferred embodiment, the monomers are chemically fused. Twomonomers are chemically fused if the two parts are chemically attached,for instance via a chemical crosslinker. Any of the chemicalcrosslinkers discussed above may be used. The linker may be attached toone or more cysteine residues introduced into a mutant monomer of theinvention. Alternatively, the linker may be attached to a terminus ofone of the monomers in the construct.

If a construct contains different monomers, crosslinkage of monomers tothemselves may be prevented by keeping the concentration of linker in avast excess of the monomers. Alternatively, a “lock and key” arrangementmay be used in which two linkers are used. Only one end of each linkermay react together to form a longer linker and the other ends of thelinker each react with a different monomers. Such linkers are describedin International Application No. PCT/GB10/000132 (published as WO2010/086602).

Mutant αHL and Lysenin Monomers

The present invention also provides mutant αHL monomers. The mutant Mspmonomers may be used to form the pores of the invention. A mutant αHLmonomer is a monomer whose sequence varies from that of a wild-type αHLmonomer and which retains the ability to form a pore. Methods forconfirming the ability of mutant monomers to form pores are well-knownin the art and are discussed in more detail below.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 4. Variants are discussed above. The mutant αHLmonomer is modified in accordance with the invention. A part of themonomer which interacts with a polynucleotide binding protein has beenmodified. The part of the monomer which interacts with a polynucleotidebinding protein when the polynucleotide binding protein is used tocontrol the movement of a target polynucleotide with respect to, orthrough, a pore comprising the monomer has been modified. Any of themodifications discussed above may be made in the monomer of theinvention.

The monomer preferably comprises a variant of SEQ ID NO: 4 whichcomprises phenylalanine (F), tryptophan (W), isoleucine (I), leucine(L), valine (V), alanine (A), arginine (R), lysine (K), aspartic acid(D), glutamic acid (E) or tyrosine (Y) at one or more of positions:

(a) 16, 17, 18, 19, 21, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242,281, 283, 285, 287, 288 and 293 or at one or more of the correspondingpositions thereof;

(b) 17, 18, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242, 287, 288and 293 or the corresponding positions thereof;

(c) 17, 19, 46, 47, 93, 236, 237, 238, 239, 240, 241, 242 and 287 or thecorresponding positions thereof; or

(d) 17, 19, 46, 93, 236, 237, 239, 240, 287 and 288 or the correspondingpositions thereof.

The present invention also provides mutant lysenin monomers. The mutantlysenin monomers may be used to form the pores of the invention. Amutant lysenin monomer is a monomer whose sequence varies from that of awild-type lysenin monomer and which retains the ability to form a pore.Methods for confirming the ability of mutant monomers to form pores arewell-known in the art and are discussed in more detail below.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 36. Variants are discussed above. The mutant lyseninmonomer is modified in accordance with the invention. A part of themonomer which interacts with a polynucleotide binding protein has beenmodified. The part of the monomer which interacts with a polynucleotidebinding protein when the polynucleotide binding protein is used tocontrol the movement of a target polynucleotide with respect to, orthrough, a pore comprising the monomer has been modified. Any of themodifications discussed above may be made in the monomer of theinvention.

The monomer preferably comprises a variant of SEQ ID NO: 36 whichcomprises phenylalanine (F), tryptophan (W), isoleucine (I), leucine(L), valine (V), alanine (A), arginine (R), lysine (K), aspartic acid(D), glutamic acid (E) or tyrosine (Y) at one or more of positions 31,33, 108, 109, 110 and 138 or the corresponding positions thereof.

The mutant αHL monomers and mutant lysenin monomers of the invention maybe used to form constructs, homo-oligomeric pores and hetero-oligomericpores in the same way as mutant Msp monomers discussed above. Such porestypically comprise seven mutant αHL monomers.

Modified Polynucleotide Binding Proteins

The method also provides a polynucleotide binding protein modified inaccordance with the invention. A part of the polynucleotide bindingprotein which interacts with a transmembrane pore has been modified. Thepart of the polynucleotide binding protein which interacts with atransmembrane pore when the polynucleotide binding protein is used tocontrol the movement of a target polynucleotide with respect to, orthrough, the pore has been modified. Any of the modifications discussedabove may be made in the proteins of the invention.

The polynucleotide binding protein preferably comprises a variant of thesequence shown in SEQ ID NO: 24. Preferred variants are discussed above.The part of the variant of SEQ ID NO: 24 that may be modified inaccordance with the invention is discussed above. The modifiedpolynucleotide binding protein of the invention preferably comprises avariant of SEQ ID NO: 24 which comprises K199A, K199V, K199F, K199D,K199S, K199W or K199L. The variant of SEQ ID NO: 24 preferably furthercomprises any of the modifications discussed below.

The polynucleotide binding protein preferably comprises a variant of thesequence shown in SEQ ID NO: 9. Preferred variants are discussed above.The part of the variant of SEQ ID NO: 9 that may be modified inaccordance with the invention is discussed above. The modifiedpolynucleotide binding protein of the invention preferably comprises avariant of SEQ ID NO: 9 which comprises phenylalanine (F), tryptophan(W), isoleucine (I), leucine (L), valine (V), alanine (A), arginine (R),lysine (K), aspartic acid (D), glutamic acid (E) or tyrosine (Y) at oneor more of positions 80, 81, 82, 84, 85, 205, 206, 209, 215, 216, 220,221, 224, 236, 240, 241, 267, 270, 272, 278, 287, 289, 293, 296, 307,308, 309, 310, 320, 321, 322, 323, 327, 349, 415, 418 and 419 or thecorresponding positions thereof, such as at one or more of:

(a) positions 80, 84, 205, 209, 215, 216, 221, 224, 236, 241, 267, 272,289, 296, 307, 308, 309, 320, 321, 322, and 419 or the correspondingpositions thereof;

(b) positions 80, 84, 209, 215, 216, 221, 267, 272, 289, 307, 308, 309,321 and 322 or the corresponding positions thereof, or

(b) positions 215, 267, 272, 307, 308 and 322 or the correspondingpositions thereof.

Polynucleotides

The present invention provides polynucleotide sequences which encode amodified transmembrane pore of the invention. The modified pore may beany of those discussed above or below.

The present invention also provides polynucleotide sequences whichencode a mutant monomer of the invention. The mutant monomer may be anyof those discussed above. The polynucleotide sequence preferablycomprises a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologousbased on nucleotide identity to the sequence of SEQ ID NO: 1 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95% nucleotide identity over a stretch of 300 or more, forexample 375, 450, 525 or 600 or more, contiguous nucleotides (“hardhomology”). Homology may be calculated as described above. Thepolynucleotide sequence may comprise a sequence that differs from SEQ IDNO: 1 on the basis of the degeneracy of the genetic code.

The present invention also provides polynucleotide sequences whichencode any of the genetically fused constructs of the invention. Thepolynucleotide preferably comprises two or more variants of the sequenceshown in SEQ ID NO: 1. The polynucleotide sequence preferably comprisestwo or more sequences having at least 50%, 60%, 70%, 80%, 90% or 95%homology to SEQ ID NO: 1 based on nucleotide identity over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95% nucleotide identity over a stretch of 600 or more, for example 750,900, 1050 or 1200 or more, contiguous nucleotides (“hard homology”).Homology may be calculated as described above.

The present invention also provides polynucleotide sequences whichencode any of the modified polynucleotide binding proteins of theinvention. The polynucleotide sequence preferably comprises a sequenceat least 50%, 60%, 70%, 80%, 90% or 95% homologous based on nucleotideidentity to the sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20,21, 22, 23, 24 or 25 over the entire sequence. There may be at least80%, for example at least 85%, 90% or 95% nucleotide identity over astretch of 300 or more, for example 375, 450, 525 or 600 or more,contiguous nucleotides (“hard homology”). Homology may be calculated asdescribed above. The polynucleotide sequence may comprise a sequencethat differs from SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23,24 or 25 on the basis of the degeneracy of the genetic code.

Polynucleotide sequences may be derived and replicated using standardmethods in the art. Chromosomal DNA encoding wild-type Msp may beextracted from a pore producing organism, such as Mycobacteriumsmegmatis. The gene encoding the pore subunit may be amplified using PCRinvolving specific primers. The amplified sequence may then undergosite-directed mutagenesis. Suitable methods of site-directed mutagenesisare known in the art and include, for example, combine chain reaction.Polynucleotides encoding a construct of the invention can be made usingwell-known techniques, such as those described in Sambrook, J. andRussell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The resulting polynucleotide sequence may then be incorporated into arecombinant replicable vector such as a cloning vector. The vector maybe used to replicate the polynucleotide in a compatible host cell. Thuspolynucleotide sequences may be made by introducing a polynucleotideinto a replicable vector, introducing the vector into a compatible hostcell, and growing the host cell under conditions which bring aboutreplication of the vector. The vector may be recovered from the hostcell. Suitable host cells for cloning of polynucleotides are known inthe art and described in more detail below.

The polynucleotide sequence may be cloned into suitable expressionvector. In an expression vector, the polynucleotide sequence istypically operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by the host cell.Such expression vectors can be used to express a pore subunit.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotidesequences may be introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a mutant monomer or construct of the invention can be produced byinserting a polynucleotide sequence into an expression vector,introducing the vector into a compatible bacterial host cell, andgrowing the host cell under conditions which bring about expression ofthe polynucleotide sequence. The recombinantly-expressed monomer orconstruct may self-assemble into a pore in the host cell membrane.Alternatively, the recombinant pore produced in this manner may beremoved from the host cell and inserted into another membrane. Whenproducing pores comprising at least two different monomers orconstructs, the different monomers or constructs may be expressedseparately in different host cells as described above, removed from thehost cells and assembled into a pore in a separate membrane, such as arabbit cell membrane.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example a tetracycline resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,ara or λ_(L) promoter is typically used.

The host cell typically expresses the monomer or construct at a highlevel. Host cells transformed with a polynucleotide sequence will bechosen to be compatible with the expression vector used to transform thecell. The host cell is typically bacterial and preferably Escherichiacoli. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3),JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express avector comprising the T7 promoter. In addition to the conditions listedabove any of the methods cited in Proc Natl Acad Sci USA. 2008 Dec. 30;105(52):20647-52 may be used to express the Msp proteins.

The invention also comprises a method of producing a mutant monomer ofthe invention or a construct of the invention. The method comprisesexpressing a polynucleotide of the invention in a suitable host cell.The polynucleotide is preferably part of a vector and is preferablyoperably linked to a promoter.

Pores

The invention also provides various pores. The movement of targetpolynucleotides with respect to, such as through, the pores of theinvention is typically more consistent. The pores preferably allow moreconsistent movement from k-mer to k-mer as the target polynucleotidemoves with respect to, such as through, the pore. The pores preferablyallow the target polynucleotide to move with respect to, such asthrough, the pore more smoothly. The pore preferably reduces the amountof stuttering associated with the movement of the target polynucleotidewith respect to, such as through, the pore. The pores preferably providemore regular or less irregular movement of the target polynucleotidewith respect to, such as through, the pore.

The noise associated with the movement of a target polynucleotide withrespect to, such as through, the pore of the invention is typicallyreduced. The pores of the invention may reduce this noise by reducingunwanted movement associated with one or more k-mers, such as eachk-mer, in the target polynucleotide. The pores of the invention mayreduce the noise associated with the current level or signature for oneor more k-mers, such as each k-mer, in the target polynucleotide.

If the target polynucleotide is double stranded, the noise associatedwith movement of the complement strand relative to the template strandis reduced and/or the movement of the complement strand relative to thetemplate strand is more consistent using the pores of the invention.This is advantageous for strand sequencing of double stranded targetpolynucleotides.

The pores of the invention are ideal for characterising, such assequencing, polynucleotide sequences because they can discriminatebetween different nucleotides with a high degree of sensitivity. Thepores can surprisingly distinguish between the four nucleotides in DNAand RNA. The pores of the invention can even distinguish betweenmethylated and unmethylated nucleotides. The base resolution of pores ofthe invention is surprisingly high. The pores show almost completeseparation of all four DNA nucleotides. The pores further discriminatebetween deoxycytidine monophosphate (dCMP) and methyl-dCMP based on thedwell time in the pore and the current flowing through the pore.

The pores of the invention can also discriminate between differentnucleotides under a range of conditions. In particular, the pores willdiscriminate between nucleotides under conditions that are favourable tothe characterising, such as sequencing, of nucleic acids. The extent towhich the pores of the invention can discriminate between differentnucleotides can be controlled by altering the applied potential, thesalt concentration, the buffer, the temperature and the presence ofadditives, such as urea, betaine and DTT. This allows the function ofthe pores to be fine-tuned, particularly when sequencing. This isdiscussed in more detail below. The pores of the invention may also beused to identify polynucleotide polymers from the interaction with oneor more monomers rather than on a nucleotide by nucleotide basis.

A pore of the invention may be isolated, substantially isolated,purified or substantially purified. A pore of the invention is isolatedor purified if it is completely free of any other components, such aslipids or other pores. A pore is substantially isolated if it is mixedwith carriers or diluents which will not interfere with its intendeduse. For instance, a pore is substantially isolated or substantiallypurified if it is present in a form that comprises less than 10%, lessthan 5%, less than 2% or less than 1% of other components, such astriblock copolymers, lipids or other pores. Alternatively, a pore of theinvention may be present in a membrane. Suitable membranes are discussedbelow.

A pore of the invention may be present as an individual or single pore.Alternatively, a pore of the invention may be present in a homologous orheterologous population of two or more pores.

Modified Pores

The invention provides a transmembrane pore in which a part of thetransmembrane pore which interacts with a polynucleotide binding proteinhas been modified. The pore may be any of those discussed above. Wherethe pore comprises one or more variants of SEQ ID NO: 2, the one or morevariants preferably do not comprise or consist of E59R, D90N, D91N,D93N, D118R, D134R and E139K.

Homo-Oligomeric Pores

The invention also provides a homo-oligomeric pore derived from Mspcomprising identical mutant monomers of the invention. Thehomo-oligomeric pore may comprise any of the mutants of the invention.The homo-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. The homo-oligomeric pore of theinvention may have any of the advantages discussed above.

The homo-oligomeric pore may contain any number of mutant monomers. Thepore typically comprises at least 7, at least 8, at least 9 or at least10 identical mutant monomers, such as 7, 8, 9 or 10 mutant monomers. Thepore preferably comprises eight or nine identical mutant monomers. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers ispreferably chemically modified as discussed above.

A preferred homo-oligomeric pore comprises eight or nine subunits eachcomprising a variant of SEQ ID NO: 2 in which L74, G75, D118 and L119have been deleted and which comprises D56N, E59R, L88N, D90N, D91N,Q126R, D134R and E139K.

Methods for making pores are discussed in more detail below.

Hetero-Oligomeric Pores

The invention also provides a hetero-oligomeric pore derived from Mspcomprising at least one mutant monomer of the invention. Thehetero-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. Hetero-oligomeric pores can be madeusing methods known in the art (e.g. Protein Sci. 2002 July;11(7):1813-24).

The hetero-oligomeric pore contains sufficient monomers to form thepore. The monomers may be of any type. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers. The pore preferably comprises eight or nine monomers.

In a preferred embodiment, all of the monomers (such as 10, 9, 8 or 7 ofthe monomers) are mutant monomers of the invention and at least one ofthem differs from the others. In a more preferred embodiment, the porecomprises eight or nine mutant monomers of the invention and at leastone of them differs from the others. They may all differ from oneanother.

The mutant monomers of the invention in the pore are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the pore are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length. The mutantmonomers of the invention in the pore preferably have the same number ofamino acids deleted from positions 72 to 82 and/or positions 111 to 121.

In another preferred embodiment, at least one of the mutant monomers isnot a mutant monomer of the invention. In this embodiment, the remainingmonomers are preferably mutant monomers of the invention. Hence, thepore may comprise 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutant monomers of theinvention. Any number of the monomers in the pore may not be a mutantmonomer of the invention. The pore preferably comprises seven or eightmutant monomers of the invention and a monomer which is not a monomer ofthe invention. The mutant monomers of the invention may be the same ordifferent.

The mutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length.

The pore may comprise one or more monomers which are not mutant monomersof the invention. MspA mutant monomers which are non mutant monomers ofthe invention include monomers comprising SEQ ID NO: 2 or a comparativevariant of SEQ ID NO: 2. At least one monomer in the pore may compriseSEQ ID NO: 2 or a comparative variant of the sequence shown in SEQ IDNO: 2 which comprises one or more of, preferably all of, D90N, D91N,D93N, D118R, D134R and E139K. At least one monomer in the pore may beany of the monomers disclosed in International Application No.PCT/GB2012/050301 (published as WO/2012/107778), including thosecomprising a comparative variant of the sequence shown in SEQ ID NO: 2which comprises G75S, G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134Rand E139K. A comparative variant of SEQ ID NO: 2 is at least 50%homologous to SEQ ID NO: 2 over its entire sequence based on amino acididentity. More preferably, the comparative variant may be at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 2 over the entire sequence.

In all the embodiments discussed above, one or more, such as 2, 3, 4, 5,6, 7, 8, 9 or 10, of the mutant monomers is preferably chemicallymodified as discussed above.

Methods for making pores are discussed in more detail below.

Construct-Containing Pores

The invention also provides a pore comprising at least one construct ofthe invention. A construct of the invention comprises two or morecovalently attached monomers derived from Msp wherein at least one ofthe monomers is a mutant monomer of the invention. In other words, aconstruct must contain more than one monomer. The pore containssufficient constructs and, if necessary, monomers to form the pore. Forinstance, an octameric pore may comprise (a) four constructs eachcomprising two constructs, (b) two constructs each comprising fourmonomers or (b) one construct comprising two monomers and six monomersthat do not form part of a construct. For instance, an nonameric poremay comprise (a) four constructs each comprising two constructs and onemonomer that does not form part of a construct, (b) two constructs eachcomprising four monomers and a monomer that does not form part of aconstruct or (b) one construct comprising two monomers and sevenmonomers that do not form part of a construct. Other combinations ofconstructs and monomers can be envisaged by the skilled person.

At least two of the monomers in the pore are in the form of a constructof the invention. The construct, and hence the pore, comprises at leastone mutant monomer of the invention. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers, in total (at least two of which must be in a construct).The pore preferably comprises eight or nine monomers (at least two ofwhich must be in a construct).

The construct containing pore may be a homo-oligomer (i.e. includeidentical constructs) or be a hetero-oligomer (i.e. where at least oneconstruct differs from the others).

A pore typically contains (a) one construct comprising two monomers and(b) 5, 6, 7 or 8 monomers. The construct may be any of those discussedabove. The monomers may be any of those discussed above, includingmutant monomers of the invention, monomers comprising SEQ ID NO: 2 andmutant monomers comprising a comparative variant of SEQ ID NO: 2 asdiscussed above.

Another typical pore comprises more than one construct of the invention,such as two, three or four constructs of the invention. If necessary,such pores further comprise sufficient additional monomers or constructsto form the pore. The additional monomer(s) may be any of thosediscussed above, including mutant monomers of the invention, monomerscomprising SEQ ID NO: 2 and mutant monomers comprising a comparativevariant of SEQ ID NO: 2 as discussed above. The additional construct(s)may be any of those discussed above or may be a construct comprising twoor more covalently attached MspA monomers each comprising a monomercomprising SEQ ID NO: 2 or a comparative variant of SEQ ID NO: 2 asdiscussed above.

A further pore of the invention comprises only constructs comprising 2monomers, for example a pore may comprise 4, 5, 6, 7 or 8 constructscomprising 2 monomers. At least one construct is a construct of theinvention, i.e. at least one monomer in the at least one construct, andpreferably each monomer in the at least one construct, is a mutantmonomer of the invention. All of the constructs comprising 2 monomersmay be constructs of the invention.

A specific pore according to the invention comprises four constructs ofthe invention each comprising two monomers, wherein at least one monomerin each construct, and preferably each monomer in each construct, is amutant monomer of the invention. The constructs may oligomerise into apore with a structure such that only one monomer of each constructcontributes to the channel of the pore. Typically the other monomers ofthe construct will be on the outside of the channel of the pore. Forexample, pores of the invention may comprise 5, 6, 7 or 8 constructscomprising 2 monomers where the channel comprises 8 monomers.

Mutations can be introduced into the construct as described above. Themutations may be alternating, i.e. the mutations are different for eachmonomer within a two monomer construct and the constructs are assembledas a homo-oligomer resulting in alternating modifications. In otherwords, monomers comprising MutA and MutB are fused and assembled to forman A-B:A-B:A-B:A-B pore. Alternatively, the mutations may beneighbouring, i.e. identical mutations are introduced into two monomersin a construct and this is then oligomerised with different mutantmonomers or constructs. In other words, monomers comprising MutA arefused followed by oligomerisation with MutB-containing monomers to formA-A:B:B:B:B:B:B.

One or more of the monomers of the invention in a construct-containingpore may be chemically-modified as discussed above.

Combinations

The invention also provides a combination of a transmembrane pore and apolynucleotide binding protein in which a part of the transmembrane porewhich interacts with the polynucleotide binding protein and/or a part ofthe polynucleotide binding protein which interacts with thetransmembrane pore has been modified. The pore may be any of thosediscussed above. The polynucleotide binding protein may be any of thosediscussed above.

The pore in the combination preferably comprises seven or more monomers,such as 8 or 9 monomers, comprising the sequence shown in SEQ ID NO: 2or a variant thereof, wherein one or more of the seven or more monomers,such as 2, 3, 4, 5, 6, 7, 8 or 9 of the monomers, comprises a variant ofSEQ ID NO: 2 which comprises (a) D56N, D56R, D56F, D56Y or D56L and/or(b) E59N, E59R, E59F, E59Y or E59L (preferably D56N and E59R, D56F andE59R, D56N and E59F, D56N and E59Y or D56L and E59L or more preferablyD56N and E59R, D56F and E59R or D56N and E59F) and the modifiedpolynucleotide binding protein in the combination preferably comprises avariant of SEQ ID NO: 24 which comprises K199A, K199V, K199F, K199D,K199S, K199W or K199L. The variant of SEQ ID NO: 24 preferably furthercomprises any of the modifications discussed below:

Preferred combinations of variants are shown in each row below.

SEQ ID NO: 2 comprising SEQ ID NO: 24 comprising D56N and E59R K199LD56F and E59R K199L D56N and E59F K199LPolynucleotide Characterisation

The invention provides a method of characterising a targetpolynucleotide. The method involves measuring one or morecharacteristics of the target polynucleotide. The target polynucleotidemay also be called the template polynucleotide or the polynucleotide ofinterest.

Polynucleotide

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the polynucleotidecan be oxidized or methylated.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide can comprise onestrand of RNA hybridised to one strand of DNA. The polynucleotide may beany synthetic nucleic acid known in the art, such as peptide nucleicacid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA),locked nucleic acid (LNA) or other synthetic polymers with nucleotideside chains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The polynucleotide can be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 400 or at least 500nucleotides or nucleotide pairs in length. The polynucleotide can be1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotidesor nucleotide pairs in length or 100000 or more nucleotides ornucleotide pairs in length.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 50, 100 or more polynucleotides. If two or morepolynucleotides are characterized, they may be different polynucleotidesor two instances of the same polynucleotide.

The polynucleotide can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of amanufactured oligonucleotide. The method is typically carried out invitro.

Sample

Each analyte is typically present in any suitable sample. The inventionis typically carried out on two or more samples that are known tocontain or suspected to contain the analytes. Alternatively, theinvention may be carried out on two or more samples to confirm theidentity of two or more analytes whose presence in the samples is knownor expected.

The first sample and/or second sample may be a biological sample. Theinvention may be carried out in vitro using at least one sample obtainedfrom or extracted from any organism or microorganism. The first sampleand/or second sample may be a non-biological sample. The non-biologicalsample is preferably a fluid sample. Examples of non-biological samplesinclude surgical fluids, water such as drinking water, sea water orriver water, and reagents for laboratory tests.

The first sample and/or second sample is typically processed prior tobeing used in the invention, for example by centrifugation or by passagethrough a membrane that filters out unwanted molecules or cells, such asred blood cells. The first sample and/or second sample may be measuredimmediately upon being taken. The first sample and/or second sample mayalso be typically stored prior to assay, preferably below −70° C.

Characterisation

The method may involve measuring two, three, four or five or morecharacteristics of the polynucleotide. The one or more characteristicsare preferably selected from (i) the length of the polynucleotide, (ii)the identity of the polynucleotide, (iii) the sequence of thepolynucleotide, (iv) the secondary structure of the polynucleotide and(v) whether or not the polynucleotide is modified. Any combination of(i) to (v) may be measured in accordance with the invention, such as{i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii},{ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv},{i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v},{ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v},{i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}. Different combinationsof (i) to (v) may be measured for the first polynucleotide compared withthe second polynucleotide, including any of those combinations listedabove.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the polynucleotide andthe pore or the duration of interaction between the polynucleotide andthe pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the polynucleotide orwithout measurement of the sequence of the polynucleotide. The former isstraightforward; the polynucleotide is sequenced and thereby identified.The latter may be done in several ways. For instance, the presence of aparticular motif in the polynucleotide may be measured (withoutmeasuring the remaining sequence of the polynucleotide). Alternatively,the measurement of a particular electrical and/or optical signal in themethod may identify the polynucleotide as coming from a particularsource.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50): 17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not thepolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcytosine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The target polynucleotide is contacted with a pore of the invention. Thepore is typically present in a membrane. Suitable membranes arediscussed below. The method may be carried out using any apparatus thatis suitable for investigating a membrane/pore system in which a pore ispresent in a membrane. The method may be carried out using any apparatusthat is suitable for transmembrane pore sensing. For example, theapparatus comprises a chamber comprising an aqueous solution and abarrier that separates the chamber into two sections. The barriertypically has an aperture in which the membrane containing the pore isformed. Alternatively the barrier forms the membrane in which the poreis present.

The method may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunnelling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11(1):279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined withelectrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January;81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO 2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International Application WO2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across themembrane. The applied potential may be a voltage potential.Alternatively, the applied potential may be a chemical potential. Anexample of this is using a salt gradient across a membrane, such as anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the currentpassing through the pore as a polynucleotide moves with respect to thepore is used to estimate or determine the sequence of thepolynucleotide. This is strand sequencing.

The method may involve measuring the current passing through the pore asthe polynucleotide moves with respect to, such as through, the pore.Therefore the apparatus used in the method may also comprise anelectrical circuit capable of applying a potential and measuring anelectrical signal across the membrane and pore. The methods may becarried out using a patch clamp or a voltage clamp. The methodspreferably involve the use of a voltage clamp.

The method of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +5 V to−5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. Thevoltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV.The voltage used is preferably in a range having a lower limit selectedfrom −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0mV and an upper limit independently selected from +10 mV, +20 mV, +50mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used ismore preferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The method is typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The charge carriers may be asymmetric acrossthe membrane. For instance, the type and/or concentration of the chargecarriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration maybe 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M,from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to1.4 M. The salt concentration is preferably from 150 mM to 1 M. Themethod is preferably carried out using a salt concentration of at least0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M orat least 3.0 M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.

The method is typically carried out in the presence of a buffer. In theexemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The method may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16 OC to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20 OC to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

Polynucleotide Binding Protein

Step (a) preferably comprises contacting the polynucleotide with apolynucleotide binding protein such that the protein controls themovement of the polynucleotide with respect to, such as through, thepore.

More preferably, the method comprises (a) contacting the polynucleotidewith the pore of the invention and a polynucleotide binding protein suchthat the protein controls the movement of the polynucleotide through thepore and (b) taking one or more measurements as the polynucleotide moveswith respect to the pore, wherein the measurements are indicative of oneor more characteristics of the polynucleotide, and therebycharacterising the polynucleotide.

More preferably, the method comprises (a) contacting the polynucleotidewith the pore of the invention and a polynucleotide binding protein suchthat the protein controls the movement of the polynucleotide through thepore and (b) measuring the current through the pore as thepolynucleotide moves with respect to the pore, wherein the current isindicative of one or more characteristics of the polynucleotide, andthereby characterising the polynucleotide.

The polynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement with respectto, or through, the pore. It is straightforward in the art to determinewhether or not a protein binds to a polynucleotide. The proteintypically interacts with and modifies at least one property of thepolynucleotide. The protein may modify the polynucleotide by cleaving itto form individual nucleotides or shorter chains of nucleotides, such asdi- or trinucleotides. The protein may modify the polynucleotide byorienting it or moving it to a specific position, i.e. controlling itsmovement. The polynucleotide binding protein is preferably a modifiedpolynucleotide binding protein of the invention.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. A polynucleotide handling enzyme is apolypeptide that is capable of interacting with and modifying at leastone property of a polynucleotide. The enzyme may modify thepolynucleotide by cleaving it to form individual nucleotides or shorterchains of nucleotides, such as di- or trinucleotides. The enzyme maymodify the polynucleotide by orienting it or moving it to a specificposition. The polynucleotide handling enzyme does not need to displayenzymatic activity as long as it is capable of binding thepolynucleotide and controlling its movement with respect to, or through,the pore. For instance, the enzyme may be modified to remove itsenzymatic activity or may be used under conditions which prevent it fromacting as an enzyme. Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Theenzyme may be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. Suitable enzymes include, but are notlimited to, exonuclease I from E. coli (SEQ ID NO: 11), exonuclease IIIenzyme from E. coli (SEQ ID NO: 13), RecJ from T. thermophilus (SEQ IDNO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17), TatDexonuclease and variants thereof. Three subunits comprising the sequenceshown in SEQ ID NO: 15 or a variant thereof interact to form a trimerexonuclease. The polymerase may be PyroPhage® 3173 DNA Polymerase (whichis commercially available from Lucigen® Corporation), SD Polymerase(commercially available from Bioron®) or variants thereof. The enzyme ispreferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variant thereof. Thetopoisomerase is preferably a member of any of the Moiety Classification(EC) groups 5.99.1.2 and 5.99.1.3. The enzyme is most preferably derivedfrom a helicase, such as Hel308 Mbu (SEQ ID NO: 18), Hel308 Csy (SEQ IDNO: 19), Hel308 Mhu (SEQ ID NO: 21), TraI Eco (SEQ ID NO: 22), XPD Mbu(SEQ ID NO: 23) or a variant thereof. Any helicase may be used in theinvention. The helicase may be or be derived from a Hel308 helicase, aRecD helicase, such as TraI helicase or a TrwC helicase, a XPD helicaseor a Dda helicase. The helicase may be any of the helicases, modifiedhelicases or helicase constructs disclosed in International ApplicationNos. PCT/GB2012/052579 (published as WO 2013/057495); PCT/GB2012/053274(published as WO 2013/098562); PCT/GB2012/053273 (published asWO2013098561); PCT/GB2013/051925 (published as WO 2014/013260);PCT/GB2013/051924 (published as WO 2014/013259); PCT/GB2013/051928(published as WO 2014/013262) and PCT/GB2014/052736 (published asWO/2015/055981).

The helicase preferably comprises the sequence shown in SEQ ID NO: 25(Trwc Cba) or a variant thereof, the sequence shown in SEQ ID NO: 18(Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24(Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 24 comprises (a) E94C and A360C or (b)E94C, A360C, C109A and C136A and then optionally (ΔM1)G1G2 (i.e.deletion of M1 and then addition G1 and G2).

Any number of helicases may be used in accordance with the invention.For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may beused. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting thepolynucleotide with two or more helicases. The two or more helicases aretypically the same helicase. The two or more helicases may be differenthelicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925 (publishedas WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736(published as WO/2015/055981).

A variant of SEQ ID NOs: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24or 25 is an enzyme that has an amino acid sequence which varies fromthat of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25and which retains polynucleotide binding ability. This can be measuredusing any method known in the art. For instance, the variant can becontacted with a polynucleotide and its ability to bind to and movealong the polynucleotide can be measured. The variant may includemodifications that facilitate binding of the polynucleotide and/orfacilitate its activity at high salt concentrations and/or roomtemperature. Variants may be modified such that they bindpolynucleotides (i.e. retain polynucleotide binding ability) but do notfunction as a helicase (i.e. do not move along polynucleotides whenprovided with all the necessary components to facilitate movement, e.g.ATP and Mg²⁺). Such modifications are known in the art. For instance,modification of the Mg²⁺ binding domain in helicases typically resultsin variants which do not function as helicases. These types of variantsmay act as molecular brakes (see below).

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11,13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferablybe at least 50% homologous to that sequence based on amino acididentity. More preferably, the variant polypeptide may be at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95%, amino acid identity over a stretch of 200 or more, forexample 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 ormore, contiguous amino acids (“hard homology”). Homology is determinedas described above. The variant may differ from the wild-type sequencein any of the ways discussed above with reference to SEQ ID NO: 2 and 4above. The enzyme may be covalently attached to the pore. Any method maybe used to covalently attach the enzyme to the pore.

A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with themutation Q594A). This variant does not function as a helicase (i.e.binds polynucleotides but does not move along them when provided withall the necessary components to facilitate movement, e.g. ATP and Mg²⁻).

In strand sequencing, the polynucleotide is translocated through thepore either with or against an applied potential. Exonucleases that actprogressively or processively on double stranded polynucleotides can beused on the cis side of the pore to feed the remaining single strandthrough under an applied potential or the trans side under a reversepotential. Likewise, a helicase that unwinds the double stranded DNA canalso be used in a similar manner. A polymerase may also be used. Thereare also possibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Any helicase may be used in the method. Helicases may work in two modeswith respect to the pore. First, the method is preferably carried outusing a helicase such that it moves the polynucleotide through the porewith the field resulting from the applied voltage. In this mode the 5′end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide into the pore such that it is passedthrough the pore with the field until it finally translocates through tothe trans side of the membrane. Alternatively, the method is preferablycarried out such that a helicase moves the polynucleotide through thepore against the field resulting from the applied voltage. In this modethe 3′ end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide through the pore such that it ispulled out of the pore against the applied field until finally ejectedback to the cis side of the membrane.

The method may also be carried out in the opposite direction. The 3′ endof the polynucleotide may be first captured in the pore and the helicasemay move the polynucleotide into the pore such that it is passed throughthe pore with the field until it finally translocates through to thetrans side of the membrane.

When the helicase is not provided with the necessary components tofacilitate movement or is modified to hinder or prevent its movement, itcan bind to the polynucleotide and act as a brake slowing the movementof the polynucleotide when it is pulled into the pore by the appliedfield. In the inactive mode, it does not matter whether thepolynucleotide is captured either 3′ or 5′ down, it is the applied fieldwhich pulls the polynucleotide into the pore towards the trans side withthe enzyme acting as a brake. When in the inactive mode, the movementcontrol of the polynucleotide by the helicase can be described in anumber of ways including ratcheting, sliding and braking. Helicasevariants which lack helicase activity can also be used in this way.

The polynucleotide may be contacted with the polynucleotide bindingprotein and the pore in any order. It is preferred that, when thepolynucleotide is contacted with the polynucleotide binding protein,such as a helicase, and the pore, the polynucleotide firstly forms acomplex with the protein. When the voltage is applied across the pore,the polynucleotide/protein complex then forms a complex with the poreand controls the movement of the polynucleotide with respect to, such asthrough, the pore.

Any steps in the method using a polynucleotide binding protein aretypically carried out in the presence of free nucleotides or freenucleotide analogues and an enzyme cofactor that facilitates the actionof the polynucleotide binding protein. The free nucleotides may be oneor more of any of the individual nucleotides discussed above. The freenucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The free nucleotides are preferably selected from AMP, TMP, GMP, CMP,UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferablyadenosine triphosphate (ATP). The enzyme cofactor is a factor thatallows the construct to function. The enzyme cofactor is preferably adivalent metal cation. The divalent metal cation is preferably Mg²⁺,Mn²⁻, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

Helicase(s) and Molecular Brake(s)

In a preferred embodiment, the method comprises:

-   -   (a) providing the polynucleotide with one or more helicases and        one or more molecular brakes attached to the polynucleotide;    -   (b) contacting the polynucleotide with a pore of the invention        and applying a potential across the pore such that the one or        more helicases and the one or more molecular brakes are brought        together and both control the movement of the polynucleotide        through the pore;    -   (c) taking one or more measurements as the polynucleotide moves        with respect to the pore wherein the measurements are indicative        of one or more characteristics of the polynucleotide and thereby        characterising the polynucleotide.

This type of method is discussed in detail in the Internationalapplication No PCT/GB2014/052737.

The one or more helicases may be any of those discussed above. The oneor more molecular brakes may be any compound or molecule which binds tothe polynucleotide and slows the movement of the polynucleotide throughthe pore. The one or more molecular brakes preferably comprise one ormore compounds which bind to the polynucleotide. The one or morecompounds are preferably one or more macrocycles. Suitable macrocyclesinclude, but are not limited to, cyclodextrins, calixarenes, cyclicpeptides, crown ethers, cucurbiturils, pillararenes, derivatives thereofor a combination thereof. The cyclodextrin or derivative thereof may beany of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J.Am. Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-3-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

The one or more molecular brakes are preferably not one or more singlestranded binding proteins (SSB). The one or more molecular brakes aremore preferably not a single-stranded binding protein (SSB) comprising acarboxy-terminal (C-terminal) region which does not have a net negativecharge or (ii) a modified SSB comprising one or more modifications inits C-terminal region which decreases the net negative charge of theC-terminal region. The one or more molecular brakes are most preferablynot any of the SSBs disclosed in International Application No.PCT/GB2013/051924 (published as WO 2014/013259).

The one or more molecular brakes are preferably one or morepolynucleotide binding proteins. The polynucleotide binding protein maybe any protein that is capable of binding to the polynucleotide andcontrolling its movement through the pore. It is straightforward in theart to determine whether or not a protein binds to a polynucleotide. Theprotein typically interacts with and modifies at least one property ofthe polynucleotide. The protein may modify the polynucleotide bycleaving it to form individual nucleotides or shorter chains ofnucleotides, such as di- or trinucleotides. The moiety may modify thepolynucleotide by orienting it or moving it to a specific position, i.e.controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. The one or more molecular brakes may bederived from any of the polynucleotide handling enzymes discussed above.Modified versions of Phi29 polymerase (SEQ ID NO: 8) which act asmolecular brakes are disclosed in U.S. Pat. No. 5,576,204. The one ormore molecular brakes are preferably derived from a helicase.

Any number of molecular brakes derived from a helicase may be used. Forinstance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used asmolecular brakes. If two or more helicases are be used as molecularbrakes, the two or more helicases are typically the same helicase. Thetwo or more helicases may be different helicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. The one or more molecular brakes derived from helicases arepreferably modified to reduce the size of an opening in thepolynucleotide binding domain through which in at least oneconformational state the polynucleotide can unbind from the helicase.This is disclosed in WO 2014/013260.

Preferred helicase constructs for use in the invention are described inInternational Application Nos. PCT/GB2013/051925 (published as WO2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

Spacers

The one or more helicases may be stalled at one or more spacers asdiscussed in International Application No. PCT/GB2014/050175. Anyconfiguration of one or more helicases and one or more spacers disclosedin the International Application may be used in this invention.

Membrane

The pore of the invention may be present in a membrane. In the method ofthe invention, the polynucleotide is typically contacted with the poreof the invention in a membrane. Any membrane may be used in accordancewith the invention. Suitable membranes are well-known in the art. Themembrane is preferably an amphiphilic layer. An amphiphilic layer is alayer formed from amphiphilic molecules, such as phospholipids, whichhave both hydrophilic and lipophilic properties. The amphiphilicmolecules may be synthetic or naturally occurring. Non-naturallyoccurring amphiphiles and amphiphiles which form a monolayer are knownin the art and include, for example, block copolymers (Gonzalez-Perez etal., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymericmaterials in which two or more monomer sub-units are polymerizedtogether to create a single polymer chain. Block copolymers typicallyhave properties that are contributed by each monomer sub-unit. However,a block copolymer may have unique properties that polymers formed fromthe individual sub-units do not possess. Block copolymers can beengineered such that one of the monomer sub-units is hydrophobic (i.e.lipophilic), whilst the other sub-unit(s) are hydrophilic whilst inaqueous media. In this case, the block copolymer may possess amphiphilicproperties and may form a structure that mimics a biological membrane.The block copolymer may be a diblock (consisting of two monomersub-units), but may also be constructed from more than two monomersub-units to form more complex arrangements that behave asamphiphiles.The copolymer may be a triblock, tetrablock or pentablock copolymer. Themembrane is preferably a triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesized, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customize polymerbased membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed inInternational Application No. PCT/GB2013/052766 (published asWO/2014/06443) or PCT/GB2013/052767(published as WO/2014/064444).

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved. Theamphiphilic layer may be supported.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁻⁸ cm s−1. This means that the pore and coupled polynucleotide cantypically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cellmembranes and serve as excellent platforms for a range of experimentalstudies. For example, lipid bilayers can be used for in vitroinvestigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Coupling

The polynucleotide is preferably coupled to the membrane comprising thepore of the invention. The method may comprise coupling thepolynucleotide to the membrane comprising the pore of the invention. Thepolynucleotide is preferably coupled to the membrane using one or moreanchors. The polynucleotide may be coupled to the membrane using anyknown method.

Each anchor comprises a group which couples (or binds) to thepolynucleotide and a group which couples (or binds) to the membrane.Each anchor may covalently couple (or bind) to the polynucleotide and/orthe membrane. If a Y adaptor and/or a hairpin loop adaptors are used,the polynucleotide is preferably coupled to the membrane using theadaptor(s).

The polynucleotide may be coupled to the membrane using any number ofanchors, such as 2, 3, 4 or more anchors. For instance, a polynucleotidemay be coupled to the membrane using two anchors each of whichseparately couples (or binds) to both the polynucleotide and membrane.

The one or more anchors may comprise the one or more helicases and/orthe one or more molecular brakes.

If the membrane is an amphiphilic layer, such as a copolymer membrane ora lipid bilayer, the one or more anchors preferably comprise apolypeptide anchor present in the membrane and/or a hydrophobic anchorpresent in the membrane. The hydrophobic anchor is preferably a lipid,fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid,for example cholesterol, palmitate or tocopherol. In preferredembodiments, the one or more anchors are not the pore.

The components of the membrane, such as the amphiphilic molecules,copolymer or lipids, may be chemically-modified or functionalised toform the one or more anchors. Examples of suitable chemicalmodifications and suitable ways of functionalising the components of themembrane are discussed in more detail below. Any proportion of themembrane components may be functionalized, for example at least 0.01%,at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or100%.

The polynucleotide may be coupled directly to the membrane. The one ormore anchors used to couple the polynucleotide to the membranepreferably comprise a linker. The one or more anchors may comprise oneor more, such as 2, 3, 4 or more, linkers. One linker may be used tocouple more than one, such as 2, 3, 4 or more, polynucleotides to themembrane.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. Thepolynucleotide may hybridise to a complementary sequence on the circularpolynucleotide linker.

The coupling may be permanent or stable. In other words, the couplingmay be such that the polynucleotide remains coupled to the membrane wheninteracting with the pore.

The coupling may be transient. In other words, the coupling may be suchthat the polynucleotide may decouple from the membrane when interactingwith the pore.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or an anchoring group can be added to thepolynucleotide. The addition of reactive groups to either end of apolynucleotide has been reported previously. The one or more anchorspreferably couple the polynucleotide to the membrane via hybridisation.Hybridisation in the one or more anchors allows coupling in a transientmanner as discussed above. The one or more anchors may comprise a singlestranded or double stranded polynucleotide. One part of the anchor maybe ligated to a single stranded or double stranded polynucleotide.Ligation of short pieces of ssDNA have been reported using T4 RNA ligaseI (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5).Alternatively, either a single stranded or double strandedpolynucleotide can be ligated to a double stranded polynucleotide andthen the two strands separated by thermal or chemical denaturation. Ifthe polynucleotide is a synthetic strand, the one or more anchors can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesised using a primer having areactive group attached to it.

Ideally, the polynucleotide is coupled to the membrane without having tofunctionalise the polynucleotide. This can be achieved by coupling theone or more anchors, such as a polynucleotide binding protein or achemical group, to the membrane and allowing the one or more anchors tointeract with the polynucleotide or by functionalizing the membrane. Theone or more anchors may be coupled to the membrane by any of the methodsdescribed herein. In particular, the one or more anchors may compriseone or more linkers, such as maleimide functionalised linkers.

In this embodiment, the polynucleotide is typically RNA, DNA, PNA, TNAor LNA and may be double or single stranded. This embodiment isparticularly suited to genomic DNA polynucleotides.

Where the one or more anchors comprise a protein, they may be able toanchor directly into the membrane without further functonalisation, forexample if it already has an external hydrophobic region which iscompatible with the membrane. Examples of such proteins include, but arenot limited to, transmembrane proteins, intramembrane proteins andmembrane proteins.

According to a preferred embodiment, the one or more anchors may be usedto couple a polynucleotide to the membrane when the polynucleotide isattached to a leader sequence which preferentially threads into thepore. Leader sequences are discussed in more detail below. Preferably,the polynucleotide is attached (such as ligated) to a leader sequencewhich preferentially threads into the pore. Such a leader sequence maycomprise a homopolymeric polynucleotide or an abasic region. The leadersequence is typically designed to hybridise to the one or more anchorseither directly or via one or more intermediate polynucleotides (orsplints). In such instances, the one or more anchors typically comprisea polynucleotide sequence which is complementary to a sequence in theleader sequence or a sequence in the one or more intermediatepolynucleotides (or splints). In such instances, the one or more splintstypically comprise a polynucleotide sequence which is complementary to asequence in the leader sequence.

Double Stranded Polynucleotide

The polynucleotide may be double stranded. If the polynucleotide isdouble stranded, the method preferably further comprises before thecontacting step ligating a bridging moiety, such as a hairpin loop, toone end of the polynucleotide. The two strands of the polynucleotide maythen be separated as or before the polynucleotide is contacted with thepore in accordance with the invention. The two strands may be separatedas the polynucleotide movement through the pore is controlled by apolynucleotide binding protein, such as a helicase, or molecular brake.This is described in International Application No. PCT/GB2012/051786(published as WO 2013/014451).

Linking and interrogating both strands on a double stranded construct inthis way increases the efficiency and accuracy of characterization.

Round the Corner Sequencing

In a preferred embodiment, a target double stranded polynucleotide isprovided with a bridging moiety, such as a hairpin loop, at one end andthe method comprises contacting the polynucleotide with the pore of theinvention such that both strands of the polynucleotide move through thepore and taking one or more measurements as the both strands of thepolynucleotide move with respect to the pore wherein the measurementsare indicative of one or more characteristics of the strands of thepolynucleotide and thereby characterising the target double strandedpolynucleotide. Any of the embodiments discussed above equally apply tothis embodiment.

Leader Sequence

Before the contacting step, the method preferably comprises attaching tothe polynucleotide a leader sequence which preferentially threads intothe pore. The leader sequence facilitates the method of the invention.The leader sequence is designed to preferentially thread into the poreof the invention and thereby facilitate the movement of polynucleotidethrough the pore. The leader sequence can also be used to link thepolynucleotide to the one or more anchors as discussed above.

Double Coupling

The method of the invention may involve double coupling of a doublestranded polynucleotide. In a preferred embodiment, the method of theinvention comprises:

(a) providing the double stranded polynucleotide with a Y adaptor at oneend and a bridging moiety adaptor, such as a hairpin loop adaptor, atthe other end, wherein the Y adaptor comprises one or more first anchorsfor coupling the polynucleotide to the membrane, wherein the bridgingmoiety adaptor comprises one or more second anchors for coupling thepolynucleotide to the membrane and wherein the strength of coupling ofthe bridging moiety adaptor to the membrane is greater than the strengthof coupling of the Y adaptor to the membrane;

(b) contacting the polynucleotide provided in step (a) with the pore theinvention such that the polynucleotide moves through the pore; and

(c) taking one or more measurements as the polynucleotide moves withrespect to the pore, wherein the measurements are indicative of one ormore characteristics of the polynucleotide, and thereby characterisingthe target polynucleotide.

This type of method is discussed in detail in International ApplicationNo. PCT/GB2015/050991.

Adding Hairpin Loops and Leader Sequences

Before provision, a double stranded polynucleotide may be contacted witha MuA transposase and a population of double stranded MuA substrates,wherein a proportion of the substrates in the population are Y adaptorscomprising the leader sequence and wherein a proportion of thesubstrates in the population are hairpin loop adaptors. The transposasefragments the double stranded polynucleotide analyte and ligates MuAsubstrates to one or both ends of the fragments. This produces aplurality of modified double stranded polynucleotides comprising theleader sequence at one end and the hairpin loop at the other. Themodified double stranded polynucleotides may then be investigated usingthe method of the invention.

These MuA based methods are disclosed in International Application No.PCT/GB2014/052505 (published as WO/2015/022544). They are also discussedin detail in International Application No. PCT/GB2015/050991.

One or more helicases may be attached to the MuA substrate Y adaptorsbefore they are contacted with the double stranded polynucleotide andMuA transposase. Alternatively, one or more helicases may be attached tothe MuA substrate Y adaptors before they are contacted with the doublestranded polynucleotide and MuA transposase.

One or more molecular brakes may be attached to the MuA substratehairpin loop adaptors before they are contacted with the double strandedpolynucleotide and MuA transposase. Alternatively, one or more molecularbrakes may be attached to the MuA substrate hairpin loop adaptors beforethey are contacted with the double stranded polynucleotide and MuAtransposase.

Uncoupling

The method of the invention may involve characterising multiple targetpolynucleotides and uncoupling of the at least the first targetpolynucleotide.

In a preferred embodiment, the invention involves characterising two ormore target polynucleotides. The method comprises:

-   -   (a) providing a first polynucleotide in a first sample;    -   (b) providing a second polynucleotide in a second sample;    -   (c) coupling the first polynucleotide in the first sample to a        membrane using one or more anchors;    -   (d) contacting the first polynucleotide with the pore of the        invention such that the polynucleotide moves through the pore;    -   (e) taking one or more measurements as the first polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the first        polynucleotide and thereby characterising the first        polynucleotide;    -   (f) uncoupling the first polynucleotide from the membrane;    -   (g) coupling the second polynucleotide in the second sample to        the membrane using one or more anchors;    -   (h) contacting the second polynucleotide with the pore of the        invention such that the second polynucleotide moves through the        pore; and    -   (i) taking one or more measurements as the second polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the second        polynucleotide and thereby characterising the second        polynucleotide.

This type of method is discussed in detail in International ApplicationNo. PCT/GB2015/050992. If one or more anchors comprise a hydrophobicanchor, such as cholesterol, the agent is preferably a cyclodextrin or aderivative thereof or a lipid. The cyclodextrin or derivative thereofmay be any of those disclosed in Eliseev, A. V., and Schneider, H-J.(1994) J. Am. Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). Any of the lipidsdisclosed herein may be used.

Modified Polynucleotides

Before characterisation, a target polynucleotide may be modified bycontacting the polynucleotide with a polymerase and a population of freenucleotides under conditions in which the polymerase forms a modifiedpolynucleotide using the target polynucleotide as a template, whereinthe polymerase replaces one or more of the nucleotide species in thetarget polynucleotide with a different nucleotide species when formingthe modified polynucleotide. The modified polynucleotide may then beprovided with one or more helicases attached to the polynucleotide andone or more molecular brakes attached to the polynucleotide. This typeof modification is described in International Application No.PCT/GB2015/050483. Any of the polymerases discussed above may be used.The polymerase is preferably Klenow or 9o North.

The template polynucleotide is contacted with the polymerase underconditions in which the polymerase forms a modified polynucleotide usingthe template polynucleotide as a template. Such conditions are known inthe art. For instance, the polynucleotide is typically contacted withthe polymerase in commercially available polymerase buffer, such asbuffer from New England Biolabs®. The temperature is preferably from 20to 37° C. for Klenow or from 60 to 75° C. for 9o North. A primer or a 3′hairpin is typically used as the nucleation point for polymeraseextension.

Characterisation, such as sequencing, of a polynucleotide using atransmembrane pore typically involves analyzing polymer units made up ofk nucleotides where k is a positive integer (i.e. ‘k-mers’). This isdiscussed in International Application No. PCT/GB2012/052343 (publishedas WO 2013/041878). While it is desirable to have clear separationbetween current measurements for different k-mers, it is common for someof these measurements to overlap. Especially with high numbers ofpolymer units in the k-mer, i.e. high values of k, it can becomedifficult to resolve the measurements produced by different k-mers, tothe detriment of deriving information about the polynucleotide, forexample an estimate of the underlying sequence of the polynucleotide.

By replacing one or more nucleotide species in the target polynucleotidewith different nucleotide species in the modified polynucleotide, themodified polynucleotide contains k-mers which differ from those in thetarget polynucleotide. The different k-mers in the modifiedpolynucleotide are capable of producing different current measurementsfrom the k-mers in the target polynucleotide and so the modifiedpolynucleotide provides different information from the targetpolynucleotide. The additional information from the modifiedpolynucleotide can make it easier to characterise the targetpolynucleotide. In some instances, the modified polynucleotide itselfmay be easier to characterise. For instance, the modified polynucleotidemay be designed to include k-mers with an increased separation or aclear separation between their current measurements or k-mers which havea decreased noise.

The polymerase preferably replaces two or more of the nucleotide speciesin the target polynucleotide with different nucleotide species whenforming the modified polynucleotide. The polymerase may replace each ofthe two or more nucleotide species in the target polynucleotide with adistinct nucleotide species. The polymerase may replace each of the twoor more nucleotide species in the target polynucleotide with the samenucleotide species.

If the target polynucleotide is DNA, the different nucleotide species inthe modified typically comprises a nucleobase which differs fromadenine, guanine, thymine, cytosine or methylcytosine and/or comprises anucleoside which differs from deoxyadenosine, deoxyguanosine, thymidine,deoxycytidine or deoxymethylcytidine. If the target polynucleotide isRNA, the different nucleotide species in the modified polynucleotidetypically comprises a nucleobase which differs from adenine, guanine,uracil, cytosine or methylcytosine and/or comprises a nucleoside whichdiffers from adenosine, guanosine, uridine, cytidine or methylcytidine.The different nucleotide species may be any of the universal nucleotidesdiscussed above.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which comprises a chemical group or atomabsent from the one or more nucleotide species. The chemical group maybe a propynyl group, a thio group, an oxo group, a methyl group, ahydroxymethyl group, a formyl group, a carboxy group, a carbonyl group,a benzyl group, a propargyl group or a propargylamine group.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which lacks a chemical group or atompresent in the one or more nucleotide species. The polymerase mayreplace the one or more of the nucleotide species with a differentnucleotide species having an altered electronegativity. The differentnucleotide species having an altered electronegativity preferablycomprises a halogen atom.

The method preferably further comprises selectively removing thenucleobases from the one or more different nucleotides species in themodified polynucleotide.

Other Characterisation Methods

In another embodiment, a polynucleotide is characterised by detectinglabelled species that are released as a polymerase incorporatesnucleotides into the polynucleotide. The polymerase uses thepolynucleotide as a template. Each labelled species is specific for eachnucleotide. The polynucleotide is contacted with a pore of theinvention, a polymerase and labelled nucleotides such that phosphatelabelled species are sequentially released when nucleotides are added tothe polynucleotide(s) by the polymerase, wherein the phosphate speciescontain a label specific for each nucleotide. The polymerase may be anyof those discussed above. The phosphate labelled species are detectedusing the pore and thereby characterising the polynucleotide. This typeof method is disclosed in European Application No. 13187149.3 (publishedas EP 2682460). Any of the embodiments discussed above equally apply tothis method.

In another embodiment, the invention also provides a method ofcharacterising a target polynucleotide, comprising

a) contacting the target polynucleotide with a transmembrane pore and apolynucleotide binding protein selected from TatD exonuclease,PyroPhage® 3173 DNA Polymerase, SD Polymerase and variants thereof suchthat the protein controls the movement of the polynucleotide withrespect to the transmembrane pore; and

c) taking one or more measurements as the polynucleotide moves withrespect to the transmembrane pore, wherein the measurements areindicative of one or more characteristics of the polynucleotide, andthereby characterising the target polynucleotide. In this embodiment,the transmembrane pore and/or the polynucleotide binding protein arepreferably not modified in accordance with the invention. Any of theembodiments discussed above with reference to characterisation ofpolynucleotides equally applies to this embodiment. The transmembranepore may be any of those discussed above.

Kits

The present invention also provides a kit for characterising a targetpolynucleotide. In one embodiment, the kit comprises a pore of theinvention and the components of a membrane. The membrane is preferablyformed from the components. The pore is preferably present in themembrane. The kit may comprise components of any of the membranesdisclosed above, such as an amphiphilic layer or a triblock copolymermembrane. The kit may further comprise a polynucleotide binding protein,preferably a modified polynucleotide binding protein of the invention.

In another embodiment, the kit comprises a modified polynucleotidebinding protein of the invention and a polynucleotide adaptor. Thepolynucleotide binding protein is preferably bound to the adaptor. Theadaptor may be any of those discussed above.

The kit may further comprise one or more anchors for coupling thepolynucleotide to the membrane.

The kit is preferably for characterising a double strandedpolynucleotide and preferably comprises a Y adaptor and a hairpin loopadaptor. The Y adaptor preferably has one or more helicases attached andthe hairpin loop adaptor preferably has one or more molecular brakesattached. The one or more helicases and/or the one or more molecularbrakes may be modified in accordance with the invention. The Y adaptorpreferably comprises one or more first anchors for coupling thepolynucleotide to the membrane, the hairpin loop adaptor preferablycomprises one or more second anchors for coupling the polynucleotide tothe membrane and the strength of coupling of the hairpin loop adaptor tothe membrane is preferably greater than the strength of coupling of theY adaptor to the membrane.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides orvoltage or patch clamp apparatus. Reagents may be present in the kit ina dry state such that a fluid sample resuspends the reagents. The kitmay also, optionally, comprise instructions to enable the kit to be usedin the method of the invention or details regarding for which organismthe method may be used.

Apparatus

The invention also provides an apparatus for characterising a targetpolynucleotide. The apparatus comprises a plurality of pores of theinvention or a plurality of combinations of the invention. The apparatusalso comprises a plurality of membranes. The plurality of pores arepreferably present in the plurality of membranes. The number of poresand membranes is preferably equal. Preferably, a single pore is presentin each membrane.

The apparatus preferably further comprises instructions for carrying outthe method of the invention. The apparatus may be any conventionalapparatus for polynucleotide analysis, such as an array or a chip. Anyof the embodiments discussed above with reference to the methods of theinvention are equally applicable to the apparatus of the invention. Theapparatus may further comprise any of the features present in the kit ofthe invention.

The apparatus is preferably set up to carry out the method of theinvention.

The apparatus preferably comprises:

a sensor device that is capable of supporting the plurality of pores andmembranes and being operable to perform polynucleotide characterisationusing the pores and membranes; and

at least one port for delivery of the material for performing thecharacterisation.

Alternatively, the apparatus preferably comprises:

a sensor device that is capable of supporting the plurality of pores andmembranes being operable to perform polynucleotide characterisationusing the pores and membranes; and

at least one reservoir for holding material for performing thecharacterisation.

The apparatus more preferably comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and membranes and being operable to perform polynucleotidecharacterising using the pores and membranes;

at least one reservoir for holding material for performing thecharacterising;

a fluidics system configured to controllably supply material from the atleast one reservoir to the sensor device; and

one or more containers for receiving respective samples, the fluidicssystem being configured to supply the samples selectively from one ormore containers to the sensor device.

The apparatus may be any of those described in International ApplicationNo. PCT/GB08/004127 (published as WO 2009/077734), PCT/GB10/000789(published as WO 2010/122293), International Application No.PCT/GB10/002206 (published as WO 2011/067559) or InternationalApplication No. PCT/US99/25679 (published as WO 00/28312).

The following Example illustrates the invention.

Example 1

This example describes the simulations which were run to investigate theinteraction betweenMspA-(G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8 (SEQ ID NO: 2with mutations G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K=MspAmutant 1) orMspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119=MspA mutant 2) with T4Dda-E94C/A360C/C109A/C136A (SEQ ID NO: 24 with mutationsE94C/A360C/C114A/C171A/C421D and then (ΔM1)G1G2)).

Simulations were performed using the GROMACS package version 4.0.5, withthe GROMOS 53a6 forcefield and the SPC water model.

The MspA mutant 1 and MspA mutant 2 models were based on the crystalstructure of MspA found in the protein data bank, accession code 1UUN.The relevant mutations were made using PyMOL, and in the case of MspAmutant 2 the residues L74/G75/D118/L119 were deleted from the barrel.The resultant pore models were then energy minimised using the steepestdescents algorithm. The T4 Dda-E94C/A360C/C109A/C136A model was based onthe Dda1993 structure found in the protein data bank, accession code3UPU. Again, relevant mutations were made using PyMOL, and the model wasenergy minimised using the steepest descents algorithm.

The T4 Dda-E94C/A360C/C109A/C136A model was then placed above MspAmutant 1 and MspA mutant 2. Three simulations were performed for the T4Dda-E94C/A360C/C109A/C136A/MspA mutant 1 and T4Dda-E94C/A360C/C109A/C136A/MspA mutant 2 systems, with the orientationof T4 Dda-E94C/A360C/C109A/C136A differing in each simulation (See FIG.1 for cartoon representations of the three different simulationorientations). The pore was placed into a lipid membrane comprising DPPCmolecules and the simulation box was solvated. Throughout thesimulation, restraints were applied to the backbone of the pore.However, the enzyme was unrestrained. The system was simulated in theNPT ensemble for 40 ns, using the Berendsen thermostat and Berendsenbarostat to 300 K.

The contacts between the enzyme and pore were analysed using bothGROMACS analysis software and also locally written code. FIGS. 2 to 5showed the amino acid residues which interacted in MspA mutant 1 (FIGS.2 and 3) and MspA mutant 2 (FIGS. 4 and 5) with the enzyme T4Dda-E94C/A360C/C109A/C136A. The tables below show the number of contactsobserved for both pore and enzyme amino acids (Table 4 shows the MspAmutant 1 amino acid contact points observed when the interactions weremeasured between MspA mutant 1 and T4 Dda-E94C/A360C/C109A/C136A, Table5 shows the T4 Dda-E94C/A360C/C109A/C136A amino acid contact pointsobserved when the interactions were measured between MspA mutant 1 andT4 Dda-E94C/A360C/C109A/C136A, Table 6 shows the MspA mutant 2 aminoacid contact points observed when the interactions were measured betweenMspA mutant 2 and T4 Dda-E94C/A360C/C109A/C136A, Table 7 shows the T4Dda-E94C/A360C/C109A/C136A amino acid contact points observed when theinteractions were measured between MspA mutant 2 and T4Dda-E94C/A360C/C109A/C136A). FIG. 6 shows which amino acids in the pore(MspA mutant 2) interacted with particular amino acids in the enzyme (T4Dda-E94C/A360C/C109A/C136A). The data obtained from the simulationsshowed that a greater number of interaction points were detected betweenMspA mutant 2 and T4 Dda-E94C/A360C/C109A/C136A than were detectedbetween MspA mutant 1 and T4 Dda-E94C/A360C/C109A/C136A.

TABLE 4 Run 1 Run 2 Run 3 Pore Amino Number Pore Amino Number Pore AminoNumber Acid of Acid of Acid of Residue Contacts Residue Contacts ResidueContacts 57 5304 56 5271 57 2068 59 4806 57 2262 136 1800 136 1515 1361697 59 1419 134 1443 139 1053 134 975 56 1402 52 720 56 817 54 382 134215 12 581 12 263 138 196 139 180 169 49 55 5 58 87 14 17 59 1 137 32 588 14 8 55 4 48 5 52 4 169 3 138 2 139 1 137 1

TABLE 5 Run 1 Run 2 Run 3 Enzyme Enzyme Enzyme Amino Number Amino NumberAmino Number Acid of Acid of Acid of Residue Contacts Residue ContactsResidue Contacts 2 5702 180 3619 255 2365 180 3644 199 2104 216 2126 1792205 202 1909 221 1027 178 1550 1 1378 227 929 227 513 4 981 351 239 4390 51 678 321 223 177 297 434 282 254 199 212 275 179 153 258 198 1 169178 101 224 177 194 75 177 84 257 137 204 58 197 71 256 115 176 56 5 19223 109 213 46 201 19 212 54 3 37 181 19 308 25 216 33 200 2 207 21 21128 6 1 350 11 202 28 228 5 224 26 210 4 223 26 319 3 191 17 304 2 199 12209 2 201 8 347 1 434 4 261 1 405 1 260 1 255 1 247 1

TABLE 6 Run 1 Run 2 Run 3 Pore Pore Pore Amino Number Amino Number AminoNumber Acid of Acid of Acid of Residue Contacts Residue Contacts ResidueContacts 59 26063 59 7271 56 9681 57 10231 57 4828 59 7422 134 6034 1693039 57 3640 136 5757 134 499 136 3160 169 3357 136 28 12 2083 56 168956 17 14 1132 137 374 54 1 134 432 58 134 14 1 54 44 14 10 12 1 169 8135 9 53 2 60 6 170 5

TABLE 7 Run 1 Run 2 Run 3 Enzyme Enzyme Enzyme Amino Number Amino NumberAmino Number Acid of Acid of Acid of Residue Contacts Residue ContactsResidue Contacts 350 7013 202 8318 199 4908 258 6277 180 3505 197 3828223 4829 179 1297 185 3158 195 4081 212 1089 198 2873 198 3990 258 617207 1998 438 3642 211 324 202 1645 260 3113 198 236 223 1559 207 2781265 57 180 1427 226 2563 260 55 209 1309 304 2489 259 37 210 1152 2002116 255 24 203 1150 227 1307 1 22 204 646 347 845 200 19 437 466 321831 300 18 200 431 422 818 203 14 211 347 318 740 261 12 405 176 415 733216 10 227 97 210 639 177 10 258 94 229 555 213 9 212 72 255 552 207 6256 68 224 492 337 2 216 55 228 461 204 2 189 42 208 395 434 1 228 22193 307 298 1 220 18 256 256 219 17

Example 2

This example describes how a helicase—T4 Dda-E94C/C109A/C136A/A360C (SEQID NO: 24 with mutations E94C/C109A/C136A/A360C) was used to control themovement of DNA construct X or Y (shown in FIGS. 7 and 8) through anumber of different MspA nanopores. All of the nanopores testedexhibited changes in current as the DNA translocated through thenanopore. The mutant nanopores tested exhibited either more consistentmovement of the target polynucleotide or reduced noise associated withthe movement of the target polynucleotide as it translocated through thenanopore or both.

Materials and Methods

Prior to setting up the experiment, DNA construct X or Y (finalconcentration 0.1 nM) was pre-incubated at room temperature for fiveminutes with T4 Dda-E94C/C109A/C136A/A360C (final concentration added tothe nanopore system 10 nM, which was provided in buffer (253 mM KCl, 50mM potassium phosphate, pH 8.0, 2 mM EDTA)). After five minutes, TMAD(100 μM final concentration added to the nanopore system) was added tothe pre-mix and the mixture incubated for a further 5 minutes. Finally,MgCl2 (2 mM final concentration added to the nanopore system), ATP (2 mMfinal concentration added to the nanopore system) and KCl (500 mM finalconcentration added to the nanopore system) were added to the pre-mix.

Electrical measurements were acquired from single MspA nanoporesinserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mMPotassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0). After achieving a single pore inserted in the block co-polymer,then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM PotassiumFerrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) wasflowed through the system to remove any excess MspA nanopores. 150 uL of500 mM KCl, 25 mM K Phosphate, pH8.0 was then flowed through the system.After 10 minutes a further 150 uL of 500 mM KCl, 25 mM K Phosphate,pH8.0 was flowed through the system and then the enzyme (T4Dda-E94C/C109A/C136A/A360C, 10 nM final concentration), DNA construct Xor Y (0.1 nM final concentration), fuel (MgCl2 2 mM final concentration,ATP 2 mM final concentration) pre-mix (150 μL total) was then flowedinto the single nanopore experimental system. The experiment was run at−120 mV and helicase-controlled DNA movement monitored.

Results

A number of different nanopores were investigated in order to determinethe effect of mutations to regions of the transmembrane pore which werethought to interact with the helicase T4 Dda-E94C/C109A/C136A/A360C. Themutant pores which were investigated are listed below with the baselinenanopore with which they were compared. A number of different parameterswere investigated in order to identify improved nanopores 1) the averagenoise of the signal (where noise is equal to the standard deviation ofall events in a strand, calculated over all strands) which in animproved nanopore would be lower than the baseline, 2) the averagecurrent range which was a measure of the spread of current levels withina signal and which in an improved nanopore would be higher than thebaseline, 3) the average signal to noise quoted in the table is thesignal to noise (average current range divided by average noise of thesignal) over all strands and in an improved nanopore would be higherthan the baseline and 4) the percentage of complement slipping forwardswhich in an improved nanopore would be lower than the baseline.

The measurement of complement slipping forwards was calculated using thefollowing procedure 1) the helicase controlled DNA movements were mappedto a model, 2) the helicase-controlled DNA movements were then subjectedto filtering, 3) the mapped helicase controlled DNA movements werechecked to ensure accurate mapping, 4) the transitions that wereclassified as a slipping forward movement of at least four consecutivenucleotides were then added together and a percentage based on the totalnumber of transitions was calculated.

In table 8 below, MspA mutant 3 (which contained the additionalmutations D56N/E59R) was compared to MspA mutant 1 (baseline). MspAmutant 3 exhibited a lower mean noise of the signal, a higher meancurrent range and a higher average signal to noise than MspA mutant 1.Therefore, the D56N/E59R mutations which were made to improve theinteraction between the nanopore and the enzyme resulted in reducednoise associated with the movement of the target polynucleotide throughthe nanopore.

In table 8 below, a number of MspA mutants 4-7 and 24 were compared toMspA mutant 1 (baseline). MspA mutants 4-7 and 24 all differed from MspAmutant 1 in that residues had been deleted and that mutations had beenmade in order to effect how the enzyme and the nanopore interacted (themutations which the nanopores had in common wereL88N/D90N/D91N/Q126R/D134R/E139K). MspA mutants 4-7 and 24 exhibited animprovement in at least one of the measured parameters (mean noise ofthe signal, median noise of the signal, mean current range, averagesignal to noise and percentage of complement slipping forwards) whencompared to MspA mutant 1. However, the measured improvements wereattributed to the combination of changes made to the nanopores (MspAmutants 4-7 and 24) e.g. deletions and the mutations which were made inorder to effect how the enzyme and the nanopore interacted.

Pore ID's

MspA mutant 1=MspA-(G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsG75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K).

MspA Mutant3=MspA-(D56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsD56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K).

MspA mutant4=MspA-((Del-L74/G75/D118/L119)E57R/E59N/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E57R/E59N/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119)

MspA mutant5=MspA-((Del-L74/G75/D118/L119)D56W/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56W/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 9.MspA mutant6=MspA-((Del-L74/G75/D118/L119)E59Y/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59Y/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 10.MspA mutant7=MspA-((Del-L74/G75/D118/L119)D56N/E59S/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59S/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119)MspA mutant24=MspA-((Del-L74/G75/D118/L119)D56N/E59W/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59W/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 20.

TABLE 8 Standard Deviation Standard Percentage Mean Median of the MeanMedian Deviation Average of Noise of Noise of Noise of Current Currentof the Signal Standard complement Pore thee the the Range Range Currentto Noise Median Deviation slipping ID Signal Signal Signal (pA) (pA)Range (S2N) S2N of S2N forwards 1 1.35 1.35 0.14 15.48 15.78 2.23 11.443 1.31 1.24 0.26 15.64 15.60 1.76 11.94 4 1.40 1.22 0.45 14.74 14.641.88 10.56 5 1.35 1.28 0.26 14.38 14.26 1.79 10.69 11.95 0.33 0.25 61.36 1.32 0.23 14.96 14.89 1.72 11.00 11.89 1.17 0.27 7 1.31 1.27 0.2314.96 14.91 1.73 11.39 11.26 0.29 0.43 24 1.38 1.32 0.26 15.16 15.091.85 10.97

In table 9 below, a number of MspA mutants 8-19 and 23 were compared toMspA mutant 2 (baseline). MspA mutants 8-19 and 23 all had the sameresidues deleted (L74/G75/D118/L119) and the following mutations(D90N/D91N/Q126R/D134R/E134K) as MspA mutant 2 but they differed fromMspA mutant 2 in the fact that they had been mutated at a range ofpositions which effected how the enzyme and the nanopore interacted. Ofthe various parameters which were investigated and measured—mean noiseof the signal, mean current range, average signal to noise andpercentage of complement slipping forwards MspA mutants 8-19 and 23exhibited an improvement in at least one of these parameters whencompared to the baseline nanopore MspA mutant 2. Therefore, themutations which were made to improve the interaction between thenanopore and the enzyme resulted either in reduced noise associated withthe movement of the target polynucleotide through the pore or moreconsistent movement of the target through the pore.

In table 9 below, a number of MspA mutants 20-22 were compared to MspAmutant 2 (baseline). MspA mutants 20-22 all differed from MspA mutant 2in the residues which had been deleted and the mutations which were madein order to effect how the enzyme and the nanopore interacted (themutations which the nanopores had in common wereL88N/D90N/D91N/Q126R/D134R/E139K). MspA mutants 20-22 exhibited animprovement in at least one of the measured parameters (mean noise ofthe signal, mean current range, average signal to noise and percentageof complement slipping forwards) when compared to MspA mutant 2.However, the measured improvements in noise and movement consistencywere attributed to the combination of changes made to the nanopores(MspA mutants 20-22) e.g. deletions and the mutations which were made inorder to effect how the enzyme and the nanopore interacted.

Pore ID's

MspA mutant2=MspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 11.MspA mutant8=MspA-((Del-L74/G75/D118/L119)D56N/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119)MspA mutant9=MspA-((Del-L74/G75/D118/L119)E59N/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59N/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119)MspA mutant10=MspA-((Del-L74/G75/D118/L119)D56N/E57N/E59N/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsD56N/E57N/E59N/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of theamino acids L74/G75/D118/L119)MspA mutant11=MspA-((Del-L74/G75/D118/L119)E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59R/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119)MspA mutant12=MspA-((Del-L74/G75/D118/L119)D56Y/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations)D56Y/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 12.MspA mutant13=MspA-((Del-L74/G75/D118/L119)D56N/E57D/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsD56N/E57D/E59R/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of theamino acids L74/G75/D118/L119). Example helicase controlled DNA movementshown in FIG. 13.MspA mutant14=MspA-((Del-L74/G75/D118/L119)D56N/E59T/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59T/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 14.MspA mutant15=MspA-((Del-L74/G75/D118/L119)D56N/E59Q/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59Q/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 15.MspA mutant16=MspA-((Del-L74/G75/D118/L119)E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations E59F/L88N/D90N/D91N/Q126R/D134R/E139K anddeletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 16.MspA mutant17=MspA-((Del-L74/G75/D118/L119)D56N/E59R/L88N/D90N/D91N/Q126R/D134N/E139K)8(SEQ ID NO: 2 with mutations D56N/E59R/L88N/D90N/D91N/Q126R/D134N/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 19.MspA mutant18=MspA-((Del-L74/G75/D118/L119)D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 17.MspA mutant19=MspA-((Del-L74/G75/D118/L119)D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119). Example helicasecontrolled DNA movement shown in FIG. 18.MspA mutant 20=MspA((Del-F80/S81/G112/V113)D56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8 (SEQ ID NO: 2 with mutationsD56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K and deletionof the amino acids F80/S81/G112/V113)MspA mutant 21=MspA((Del-G75/V76/A117/D118)D56N/E59R/G77S/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsD56N/E59R/G77S/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of theamino acids G75/V76/A117/D118)MspA mutant22=MspA-((Del-N79/F80/V113/V14)D56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8 (SEQ ID NO: 2 with mutationsD56N/E59R/G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K and deletionof the amino acids N79/F80/V113/V114)MspA mutant23=MspA-((Del-L74/G75/D118/L119)D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119).

TABLE 9 Standard Standard Deviation Deviation Percentage Mean Median ofthe Mean Median of the Average of Noise of Noise of Noise of CurrentCurrent Current Signal Standard complement Pore the the the Range RangeRange to Noise Median Deviation slipping ID Signal Signal Signal (pA)(pA) (pA) (S2N) S2N of S2N forwards 2 1.22 1.17 0.20 15.31 15.32 1.6612.50 0.49 8 1.20 1.17 0.15 14.74 14.62 1.66 12.28 9 1.11 1.08 0.1914.74 14.71 2.36 13.28 10 1.46 1.35 0.36 19.02 19.16 2.07 13.03 11 1.261.22 0.24 16.08 16.16 1.84 12.76 12 1.24 1.19 0.22 15.93 15.92 1.8312.88 13.34 1.12 0.60 13 1.19 1.16 0.19 15.45 15.36 1.77 12.95 11.950.33 0.70 14 1.31 1.26 0.24 16.04 16.09 1.87 12.29 12.61 0.87 0.80 151.22 1.16 0.25 15.55 15.53 1.72 12.76 13.33 0.86 0.55 16 1.37 1.33 0.2215.68 15.68 1.81 11.44 11.79 0.64 0.30 17 1.31 1.27 0.21 16.04 16.051.93 12.25 12.03 0.57 0.65 18 1.24 1.17 0.27 15.77 15.66 1.86 12.7612.76 0.12 0.25 19 1.33 1.28 0.24 15.61 15.60 1.92 11.74 12.29 0.59 0.3820 1.32 1.22 0.32 17.11 17.15 2.11 12.96 21 1.27 1.21 0.24 15.81 15.841.69 12.48 22 1.21 1.13 0.28 15.82 15.82 1.90 13.13 23 1.48 1.43 0.2415.90 15.93 1.92 10.72 10.37 0.66 0.30

Example 3

This example describes the simulations which were run to investigate theinteraction between α-hemolysin-(E111N/K147N)8 (SEQ ID NO: 4) with Phi29DNA polymerase-(D12A/D66A) (SEQ ID NO: 9 with mutations D12A/D66A).

Simulations were performed using the GROMACS package version 4.0.5, withthe GROMOS 53a6 forcefield and the SPC water model.

The αHL-(E111N/K147N)8 model was based on the crystal structure of αHLwild-type found in the protein data bank, accession code 7AHL. Therelevant mutations were made using PyMOL and the resultant pore modelwas then energy minimised using the steepest descents algorithm. ThePhi29 DNA polymerase-(D12A/D66A) (SEQ ID NO: 9 with mutations D12A/D66A)model was based on the crystal structure of Phi29 DNApolymerase-(D12A/D66A) found in the protein data bank, accession code2PYL.

The Phi29 DNA polymerase-(D12A/D66A) model was then placed aboveαHL-(E111N/K147N)8. Three simulations were performed for the Phi29 DNApolymerase-(D12A/D66A)/αHL-(E111N/K147N)8 system, with the orientationof Phi29 DNA polymerase-(D12A/D66A) differing in each simulation (SeeFIG. 21 for cartoon representations of the three different simulationorientations). The pore was placed into a lipid membrane comprising DPPCmolecules and the simulation box was solvated. Throughout thesimulation, restraints were applied to the backbone of the pore.However, the enzyme was unrestrained. The system was simulated in theNPT ensemble for 40 ns, using the Berendsen thermostat and Berendsenbarostat to 300 K.

The contacts between the enzyme and pore were analysed using bothGROMACS analysis software and also locally written code. FIGS. 22 and 23show the amino acid residues which interacted in αHL-(E111N/K147N)8 withPhi29 DNA polymerase-(D12A/D66A). The tables below show the number ofcontacts observed for both pore and enzyme amino acids (Table 10 showsthe αHL-(E111N/K147N)8 amino acid contact points observed when theinteractions were measured between αHL-(E111N/K147N)8 and Phi29 DNApolymerase-(D12A/D66A), Table 11 shows the Phi29 DNApolymerase-(D12A/D66A) amino acid contact points observed when theinteractions were measured between αHL-(E111N/K147N)8 and Phi29 DNApolymerase-(D12A/D66A). Table 10 shows all the amino acids residues inαHL-E111N/K147N)8 that made more than 100 contacts with Phi29 DNApolymerase-(D12A/D66A) and Table 11 shows all the amino acid residues inPhi29 DNA polymerase-(D12A/D66A) that made more than 100 contacts withαHL-(E111N/K147N)8. FIGS. 24-28 show which amino acids in the pore(αHL-(E111N/K147N)8) interacted with particular amino acids in theenzyme (Phi29 DNA polymerase-(D12A/D66A) in runs 1-3.

TABLE 10 Run 1 Run 2 Run 3 Pore Pore Pore Amino Number Amino NumberAmino Number Acid of Acid of Acid of Residue Contacts Residue ContactsResidue Contacts S239 3802 T19 5932 S239 6420 N93 2785 N17 5125 E2874255 K240 1217 K240 2679 K237 2618 Q242 1159 K46 2481 R236 2568 K237 958E287 2243 A238 1796 K288 706 Q241 1802 K240 1791 E287 337 N47 1496 Q2421032 D285 107 T18 535 N293 967 S239 442 K288 775 K21 398 Q241 609 K288361 R281 256 S16 163 K283 118 K237 136 N17 112

TABLE 11 Run 1 Run 2 Enzyme Enzyme Run 3 Amino Number Amino NumberEnzyme Number Acid of Acid of Acid of Residue Contacts Residue ContactsResidue Contacts E322 4107 E272 6820 R308 9172 F309 1675 E267 3135 E2725732 G321 1642 S215 2941 S307 2304 R289 798 E221 1778 F309 1409 G320 764D84 1738 R289 1064 E241 697 L216 1346 E221 699 R236 528 K209 1126 E296643 K240 420 K80 1048 Y224 511 G323 359 E419 788 E322 363 R308 180 K205633 E293 308 E418 463 H287 275 V270 383 W327 166 G85 213 K220 148 R415177 S349 129 W81 167 E418 113 D278 147 Y310 108 S82 122 K206 107

Example 4

This example describes how a number of different helicases were used tocontrol the movement of DNA construct X (see FIG. 7) through a number ofdifferent MspA nanopores. All of the nanopores tested exhibited changesin current as the DNA translocated through the nanopore. This exampleinvestigates the number of slips forward per kilobase and the % basesmissed in construct X for a number of pore/enzyme combinations. Thehelicases investigated in the example moved along the polynucleotide ina 5′ to 3′ direction. When the 5′ end of the polynucleotide (the endaway from which the helicase moves) was captured by the pore, thehelicase worked with the direction of the field resulting from theapplied potential and moved the threaded polynucleotide into the poreand into the trans chamber. In this Example, slipping forward involvedthe DNA moving forwards relative to the pore (i.e. towards its 3′ andaway from it 5′ end) at least 4 consecutive nucleotides.

Materials and Methods

Prior to setting up the experiment, DNA construct X (final concentration0.1 nM) was pre-incubated at room temperature for five minutes with theappropriate enzyme (either T4 Dda-E94C/C109A/C136A/A360C or T4Dda-E94C/C109A/C136A/K199L/A360C (final concentration added to thenanopore system 10 nM, which was provided in buffer (253 mM KCl, 50 mMpotassium phosphate, pH 8.0, 2 mM EDTA)). After five minutes, TMAD (100μM final concentration added to the nanopore system) was added to thepre-mix and the mixture incubated for a further 5 minutes. Finally,MgCl2 (2 mM final concentration added to the nanopore system), ATP (2 mMfinal concentration added to the nanopore system) and KCl (500 mM finalconcentration added to the nanopore system) were added to the pre-mix.

Electrical measurements were acquired from single MspA nanoporesinserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mMPotassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0) as described in Example 2. The appropriate MspA nanopore wasselected from the list below (MspA mutants 2, 19, 25, 26 or 27).

Results

A number of different nanopores/enzyme combinations were investigated inorder to determine the affect of mutations to regions of thetransmembrane pore and enzyme which were thought to interact with eachother. These mutation positions were identified in the molecularmodeling experiment described in Example 1. Two different parameterswere investigated in order to identify pore and enzyme combinationswhich exhibited improved helicase controlled DNA translocation 1) thenumber of slips forward per kilobase and 2) the % bases missed inconstruct X.

The measurement of slips forward per kilobase was calculated using thefollowing procedure 1) the helicase controlled DNA movements were mappedto a model, 2) the helicase-controlled DNA movements were then subjectedto filtering, 3) the mapped helicase controlled DNA movements werechecked to ensure accurate mapping, 4) the transitions that wereclassified as a slipping forward movement of at least four consecutivenucleotides were determined per kilobase. The % bases missed inconstruct X is a measure of the number of bases in construct X which aremissed as a result of slips forward along DNA construct X expressed as apercentage.

Table 12 below shows the different pore and enzyme combinations tested,the corresponding figure number which shows a number of example currenttraces when the helicase controlled the movement of construct X throughthe nanopore and the appropriate column reference for FIG. 36 whichshows the data relating to 1) the number of slips forward per kilobaseand 2) the % bases missed in construct X. All of the pore/enzymecombinations show less than 5 slips forward per kilobase and less than12% bases missed in construct X. However, the combination of MspA mutant26 with T4 Dda-E94C/C109A/C136A/K199L/A360C produced the lowest slipsforward per kilobase and the lowest % bases missed in construct X.Therefore, this was a particularly preferred combination which waspredicted from the modeling experiment in Example 1 to produce a poreand enzyme with a particularly favourable interaction and moreconsistent movement.

Pore ID's

MspA mutant25=MspA-((Del-L74/G75/D118/L119)D56L/E59L/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56L/E59L/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119)

MspA mutant26=MspA-((Del-L74/G75/D118/L119)G1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutationsG1A/D56N/E59F/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of the aminoacids L74/G75/D118/L119).

MspA mutant27=MspA-((Del-L74/G75/D118/L119)D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139K)8(SEQ ID NO: 2 with mutations D56N/E59Y/L88N/D90N/D91N/Q126R/D134R/E139Kand deletion of the amino acids L74/G75/D118/L119).

TABLE 12 FIGURE Column in Showing FIG. 36 Example Corresponding Currentto this Nanopore Enzyme Trace Combination MspA T4 Dda- 29 1 Mutant 2E94C/C109A/C136A/A360C MspA T4 Dda- 30 2 Mutant 19E94C/C109A/C136A/A360C MspA T4 Dda- 31 3 Mutant 19E94C/C109A/C136A/K199L/A360C MspA T4 Dda- 32 4 Mutant 25E94C/C109A/C136A/A360C MspA T4 Dda- 33 5 Mutant 26E94C/C109A/C136A/A360C MspA T4 Dda- 34 6 Mutant 26E94C/C109A/C136A/K199L/A360C MspA T4 Dda- 35 7 Mutant 27E94C/C109A/C136A/A360C

The invention claimed is:
 1. A method of characterizing a targetpolynucleotide, comprising: (a) providing a transmembrane pore and apolynucleotide binding protein in which a part of the polynucleotidebinding protein which interacts with the transmembrane pore has beenmodified, wherein the modification comprises an amino acid substitution,insertion, or deletion relative to an unmodified polynucleotide bindingprotein; (b) contacting the transmembrane pore and polynucleotidebinding protein provided in (a) with the target polynucleotide such thatthe polynucleotide binding protein controls the movement of thepolynucleotide with respect to the transmembrane pore; and (c) takingone or more electrical or optical measurements as the polynucleotidemoves with respect to the transmembrane pore.
 2. The method according toclaim 1, wherein the surface of the polynucleotide binding protein whichinteracts with the transmembrane pore has been modified.
 3. The methodaccording to claim 1, wherein the modification(s) alter the charge,sterics, hydrogen bonding, 7C stacking, or structure of the part of thepolynucleotide binding protein which interacts with the transmembranepore.
 4. The method according to claim 1, wherein the polynucleotidebinding protein is a helicase.
 5. The method according to claim 1,wherein the polynucleotide binding protein is a He1308 helicase, a RecDhelicase, a TraI helicase, a TrwC helicase, a XPD helicase, or a Ddahelicase.
 6. The method according to claim 1, wherein the polynucleotidebinding protein is a Dda helicase.
 7. The method according to claim 1,wherein the polynucleotide binding protein comprises the sequence shownin SEQ ID NO:
 24. 8. The method according to claim 7, wherein the partof the polynucleotide binding protein which interacts with thetransmembrane pore comprises the amino acids at (a) positions 1, 2, 3,4, 5, 6, 51, 176, 177, 178, 179, 180, 181, 185, 189, 191, 193, 194, 195,197, 198, 199, 200, 201, 202, 203, 204, 207, 208, 209, 210, 211, 212,213, 216, 219, 220, 221, 223, 224, 226, 227, 228, 229, 247, 254, 255,256, 257, 258, 259, 260, 261, 298, 300, 304, 308, 318, 319, 321, 337,347, 350, 351, 405, 415, 422, 434, 437, and 438 in SEQ ID NO: 24; (b)positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198, 199,200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223, 224,226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321, 347,350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24; or (c)positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202, 203,207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304, 350 and438 in SEQ ID NO:
 24. 9. The method according to claim 7, wherein thepolynucleotide binding protein comprises one of the following sets ofamino acid substitutions relative to SEQ ID NO: 24: (a) E94C and A360C;or (b) E94C, A360C, C109A and C136A.
 10. The method according to claim1, further wherein a part of the transmembrane pore which interacts withthe polynucleotide binding protein has been modified, wherein themodification comprises an amino acid substitution, insertion, ordeletion relative to an unmodified transmembrane pore.
 11. A method ofcharacterizing a target polynucleotide, comprising: (a) providing atransmembrane pore and a Dda helicase in which a part of the Ddahelicase which interacts with the transmembrane pore has been modified,wherein the modification comprises an amino acid substitution,insertion, or deletion relative to an unmodified Dda helicase; (b)contacting the transmembrane pore and the Dda helicase provided in (a)with the target polynucleotide such that the Dda helicase controls themovement of the polynucleotide with respect to the transmembrane pore;and (c) taking one or more electrical or optical measurements as thepolynucleotide moves with respect to the transmembrane pore.
 12. Themethod according to claim 11, wherein the Dda helicase comprises thesequence shown in SEQ ID NO:
 24. 13. The method according to claim 12,wherein the part of the Dda helicase which interacts with thetransmembrane pore comprises the amino acids at (a) positions 1, 2, 3,4, 5, 6, 51, 176, 177, 178, 179, 180, 181, 185, 189, 191, 193, 194, 195,197, 198, 199, 200, 201, 202, 203, 204, 207, 208, 209, 210, 211, 212,213, 216, 219, 220, 221, 223, 224, 226, 227, 228, 229, 247, 254, 255,256, 257, 258, 259, 260, 261, 298, 300, 304, 308, 318, 319, 321, 337,347, 350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24; (b)positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198, 199,200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223, 224,226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321, 347,350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24; or (c)positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202, 203,207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304, 350 and438 in SEQ ID NO:
 24. 14. The method according to claim 12, wherein thepolynucleotide binding protein comprises one of the following sets ofamino acid substitutions relative to SEQ ID NO: 24: (a) E94C and A360C;or (b) E94C, A360C, C109A and C136A.
 15. The method according to claim11, further wherein a part of the transmembrane pore which interactswith the Dda helicase has been modified, wherein the modificationcomprises an amino acid substitution, insertion, or deletion relative toan unmodified transmembrane pore.
 16. A method of characterizing atarget polynucleotide, comprising: (a) providing a transmembrane poreand a polynucleotide binding protein in which a part of thepolynucleotide binding protein which interacts with the transmembranepore has been modified and a part of the transmembrane pore whichinteracts with the polynucleotide binding protein has been modified,wherein the modification of the polynucleotide binding protein comprisesan amino acid substitution, insertion, or deletion relative to anunmodified polynucleotide binding protein, and wherein the modificationof the transmembrane pore comprises an amino acid substitution,insertion, or deletion relative to an unmodified transmembrane pore; (b)contacting the transmembrane pore and polynucleotide binding proteinprovided in (a) with the target polynucleotide such that thepolynucleotide binding protein controls the movement of thepolynucleotide with respect to the transmembrane pore; and (c) takingone or more electrical or optical measurements as the polynucleotidemoves with respect to the transmembrane pore.
 17. The method accordingto claim 16, wherein the polynucleotide binding protein is a helicase.18. The method according to claim 16, wherein the polynucleotide bindingprotein is a He1308 helicase, a RecD helicase, a TraI helicase, a TrwChelicase, a XPD helicase, or a Dda helicase.
 19. The method according toclaim 16, wherein the polynucleotide binding protein comprises thesequence shown in SEQ ID NO:
 24. 20. The method according to claim 19,wherein the polynucleotide binding protein comprises one of thefollowing sets of amino acid substitutions relative to SEQ ID NO: 24:(a) E94C and A360C; or (b) E94C, A360C, C109A and C136A.